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This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of β 2 -adrenoceptor (β 2 AR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSS cis (photostationary state for cis isomer) percentage (∼90 %) and a favourable half-life (>10 days), facilitating diverse bioassay measurements. In vitro, the cis-isomer displayed substantially higher β 2 AR binding affinity than the trans-isomer (1000-fold), making (S)-Opto-prop-2 one of the best photoswitchable GPCR (G protein-coupled receptor) ligands reported so far. Molecular docking of (S)-Opto-prop-2 in the X-ray structure of propranolol-bound β 2 AR followed by site-directed mutagenesis studies, identified D113 3.32 , N312 7.39 and F289 6.51 as crucial residues that contribute to ligand-receptor interactions at the molecular level. In vivo efficacy was assessed using a rabbit ocular hypertension model, revealing that the cis isomer mimicked propranolol's effects in reducing intraocular pressure, while the trans isomer was inactive. Dynamic optical modulation of β 2 AR by (S)-Opto-prop-2 was demonstrated in two different cAMP bioassays and using live-cell confocal imaging, indicating reversible and dynamic control of β 2 AR activity using the new photopharmacology tool. In conclusion, (S)-Opto-prop-2 emerges as a promising photoswitchable ligand for precise and reversible β 2 AR modulation with light. The new tool shows superior cis-on binding affinity, one of the largest reported differences in affinity (1000-fold) between its two configurations, in vivo efficacy, and dynamic modulation. This study contributes valuable insights into the evolving field of photopharmacology, offering a potential avenue for targeted therapy in β 2 AR-associated pathologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Alternative splicing significantly enhances the diversity of the G protein-coupled receptor (GPCR) family, including the histamine H 3 receptor (H 3 R). This post-transcriptional modification generates multiple H 3 R isoforms with potentially distinct pharmacological and physiological profiles. H 3 R is primarily involved in the presynaptic inhibition of neurotransmitter release in the central nervous system. Despite the approval of pitolisant for narcolepsy (Wakix ® ) and daytime sleepiness in adults with obstructive sleep apnea (Ozawade ® ) and ongoing clinical trials for other H 3 R antagonists/inverse agonists, the functional significance of the numerous H 3 R isoforms remains largely enigmatic. Recent publicly available RNA sequencing data have confirmed the expression of multiple H 3 R isoforms in the brain, with some isoforms exhibiting unique tissue-specific distribution patterns hinting at isoform-specific functions and interactions within neural circuits. In this review, we discuss the complexity of H 3 R isoforms with a focus on their potential roles in central nervous system (CNS) function. Comparative analysis across species highlights evolutionary conservation and divergence in H 3 R splicing, suggesting species-specific regulatory mechanisms. Understanding the functionality of H 3 R isoforms is crucial for the development of targeted therapeutics. This knowledge will inform the design of more precise pharmacological interventions, potentially enhancing therapeutic efficacy and reducing adverse effects in the treatment of neurological and psychiatric disorders.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

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 H 3 receptors (H 3 R) and H 4 receptors (H 4 R). Employing the photoremovable group BODIPY as the caging entity for two agonist scaffolds-immepip and 4-methylhistamine-for H 3 R and H 4 R, 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 H 3 R 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 H 3 R receptor pharmacology.

The histamine H 3 receptor (H 3 R) regulates as a presynaptic G protein-coupled receptor the release of histamine and other neurotransmitters in the brain, and is consequently a potential therapeutic target for neuronal disorders. The human H 3 R encodes for seven splice variants that vary in the length of intracellular loop 3 and/or the C-terminal tail but are all able to induce heterotrimeric G i protein signaling. The last two decades H 3 R drug discovery and lead optimization has been exclusively focused on the 445 amino acids-long reference isoform H 3 R-445. In this study, we pharmacologically characterized for the first time all seven H 3 R isoforms by determining their binding affinities for reference histamine H 3 receptor agonists and inverse agonists. The H 3 R-453, H 3 R-415, and H 3 R-413 isoforms display similar binding affinities for all ligands as the H 3 R-445. However, increased agonist binding affinities were observed for the three shorter isoforms H 3 R-329, H 3 R-365, and H 3 R-373, whereas inverse agonists such as the approved anti-narcolepsy drug pitolisant (Wakix®) displayed significantly decreased binding affinities for the latter two isoforms. This opposite change in binding affinity of agonist versus inverse agonists on H 3 R-365 and H 3 R-373 is associated with their higher constitutive activity in a cAMP biosensor assay as compared to the other five isoforms. The observed differences in pharmacology between longer and shorter H 3 R isoforms should be considered in future drug discovery programs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Meichun Gao reports financial support was provided by CSC Chinese scholarship grant. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The histamine H 4 receptor (H 4 R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H 4 R remains elusive. Here, we report four cryo-EM structures of H 4 R/G i complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D94 3.32 and a π-π network determine the orientation of the positively charged group of ligands, while E182 5.46 , located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H 4 R ligand binding allows us to identify mutants at E182 5.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and G i engagement, we establish a framework for understanding H 4 R signaling and provide a rational basis for designing novel antihistamines targeting H 4 R., (© 2024. The Author(s).)

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this β -arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC 50 = 8.3) to human ACKR3, as measured in [ 125 I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based β -arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC 50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([ 3 H]VUF15485), binds ACKR3 saturably and with high affinity ( K d = 8.2 nM). Additionally, [ 3 H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [ 3 H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [ 3 H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central and peripheral nervous systems. Because the ability to directly measure extracellular HA in real time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G-protein-coupled receptor-activation-based (GRAB) HA (GRAB HA ) sensors with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRAB HA sensors, we measured electrical-stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA dynamics between these specific brain regions. Thus, GRAB HA sensors are robust tools for measuring extracellular HA transmission in both physiological and pathological processes., Competing Interests: Declaration of interests Y.L. has filed patent applications, the value of which might be affected by this publication., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Histamine H 3 receptor (H 3 R) agonists without an imidazole moiety remain very scarce. Of these, ZEL-H16 (1) has been reported previously as a high-affinity non-imidazole H 3 R (partial) agonist. Our structure-activity relationship analysis using derivatives of 1 identified both basic moieties as key interaction motifs and the distance of these from the central core as a determinant for H 3 R affinity. However, in spite of the reported H 3 R (partial) agonism, in our hands, 1 acts as an inverse agonist for Gα i signaling in a CRE-luciferase reporter gene assay and using an H 3 R conformational sensor. Inverse agonism was also observed for all of the synthesized derivatives of 1. Docking studies and molecular dynamics simulations suggest ionic interactions/hydrogen bonds to H 3 R residues D114 3.32 and E206 5.46 as essential interaction points., (© 2022 The Authors. Archiv der Pharmazie published by Wiley-VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors β 1 -AR and β 2 -AR, applying an azologization strategy to the first-generation beta-blocker propranolol. The resulting compounds (Opto-prop-1, -2, -3) have good photochemical properties with high levels of light-induced trans - cis isomerization (>94%) and good thermal stability ( t 1/2  > 10 days) of the resulting cis -isomer in an aqueous buffer. Upon illumination with 360-nm light to PSS cis , large differences in binding affinities were observed for photoswitchable compounds at β 1 -AR as well as β 2 -AR. Notably, Opto-prop-2 (VUF17062) showed one of the largest optical shifts in binding affinities at the β 2 -AR (587-fold, cis -active), as recorded so far for photoswitches of G protein-coupled receptors. We finally show the broad utility of Opto-prop-2 as a light-dependent competitive antagonist of the β 2 -AR as shown with a conformational β 2 -AR sensor, by the recruitment of downstream effector proteins and functional modulation of isolated adult rat cardiomyocytes., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

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 H 3 receptor (H 3 R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H 3 R ligands. In the current study, we have further characterized this H 3 R 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 H 3 R 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 H 3 R 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.

The histamine H 1 receptor (H 1 R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H 1 R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H 1 R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H 1 R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H 1 R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a , both potent and subtype-selective, enabled H 1 R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H 1 R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

The field of photopharmacology of Class A GPCR ligands has recently attracted attention. In this review we analyze 31 papers on currently available photoswitchable ligands for Class A GPCRs. Using the six most recurring terms of all combined paper abstracts, one can extract the overarching goal of this area of research: "Photoswitchable ligands control receptor activity with light" (represented in the TOC graphic). We analyze the design, photochemistry and pharmacology of the photoswitchable ligands. Trends, challenges and limitations will be discussed. A number of efficient photoswitchable ligands that allow optical modulation of GPCR function in various in vitro assays are presented. Moreover, optical modulation of in vivo GPCR function is within reach and the first reports to this end are highlighted., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The histamine H 1 receptor (H 1 R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H 1 R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H 1 R signals via heterotrimeric G q proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H 1 R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H 1 R signaling through heterotrimeric G q proteins, second messengers (inositol 1,4,5-triphosphate and Ca 2+ ), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H 1 R over G q biosensor activation.

The histamine research community has in the last decade been very active and generated a number of exciting new chemical biology tools for the study of histamine receptors, their ligands, and their pharmacology. In this paper we describe the development of histamine receptor structural biology, the use of receptor conformational biosensors, and the development of new ligands for covalent or fluorescent labeling or for photopharmacological approaches (photocaging and photoswitching). These new tools allow new approaches to study histamine receptors and hopefully will lead to better insights in the molecular aspects of histamine receptors and their ligands., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The histamine H 4 receptor (H 4 R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H 4 R activates G proteins and recruits β-arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H 4 R interactors in a Jurkat T cell line cDNA library. Forty-three novel H 4 R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H 4 R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H 4 R and TSPAN4, but TSPAN4 did not affect H 4 R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.

There is an increasing interest in guiding hit optimization by considering the target binding kinetics of ligands. However, compared to conventional structure-activity relationships, structure-kinetics relationships have not been as thoroughly explored, even for well-studied archetypical drug targets such as the histamine H 1 receptor (H 1 R), a member of the family A G-protein coupled receptor. In this study, we show that the binding kinetics of H 1 R antagonists at the H 1 R is dependent on the cyclicity of both the aromatic head group and the amine moiety of H 1 R ligands, the chemotypes that are characteristic for the first-generation H 1 R antagonists. Fusing the two aromatic rings of H 1 R ligands into one tricyclic aromatic head group prolongs the H 1 R residence time for benchmark H 1 R ligands as well as for tailored synthetic analogues. The effect of constraining the aromatic rings and the basic amines is systematically explored, leading to a coherent series and detailed discussions of structure-kinetics relationships. This study shows that cyclicity has a pronounced effect on the binding kinetics., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The Exome Aggregation Consortium has collected the protein-encoding DNA sequences of almost 61,000 unrelated humans. Analysis of this dataset for G protein-coupled receptor (GPCR) proteins (available at GPCRdb) revealed a total of 463 naturally occurring genetic missense variations in the histamine receptor family. In this research, we have analyzed the distribution of these missense variations in the four histamine receptor subtypes concerning structural segments and sites important for GPCR function. Four missense variants R127 3.52×52 H, R139 34.57×57 H, R409 6.29×29 H, and E410 6.30×30 K, were selected for the histamine H 1 receptor (H 1 R) that were hypothesized to affect receptor activity by interfering with the interaction pattern of the highly conserved D(E)RY motif, the so-called ionic lock. The E410 6.30×30 K missense variant displays higher constitutive activity in G protein signaling as compared to wild-type H 1 R, whereas the opposite was observed for R127 3.52×52 H, R139 34.57×57 H, and R409 6.29×29 H. The E410 6.30×30 K missense variant displays a higher affinity for the endogenous agonist histamine than wild-type H 1 R, whereas antagonist affinity was not affected. These data support the hypothesis that the E410 6.30×30 K mutation shifts the equilibrium towards active conformations. The study of these selected missense variants gives additional insight into the structural basis of H 1 R activation and, moreover, highlights that missense variants can result in pharmacologically different behavior as compared to wild-type receptors and should consequently be considered in the drug discovery process.

Background : The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods : We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results : Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T 352 and in part S 355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

Cytosolic β-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or β-arrestin recruitment makes β-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor β-arrestin2 recruitment to the human histamine H 1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H 1 receptor.

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for G s , G q , and G i signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.

The histamine H 3 receptor (H 3 R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H 3 R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H 3 R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H 3 R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor's real-time conformational dynamics and provides an advanced tool to screen for both H 3 R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H 3 receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H 3 receptor ligand possesses even stronger inverse agonistic activity than reference H 3 R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H 3 R conformational biosensor that will aid in the discovery of novel H 3 R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target.

The histamine H 4 receptor (H 4 R) activates Gα i -mediated signaling and recruits β-arrestin2 upon stimulation with histamine. β-Arrestins play a regulatory role in G protein-coupled receptor (GPCR) signaling by interacting with phosphorylated serine and threonine residues in the GPCR C-terminal tail and intracellular loop 3, resulting in receptor desensitization and internalization. Using bioluminescence resonance energy transfer (BRET)-based biosensors, we show that G protein-coupled receptor kinases (GRK) 2 and 3 are more quickly recruited to the H 4 R than β-arrestin1 and 2 upon agonist stimulation, whereas receptor internalization dynamics toward early endosomes was slower. Alanine-substitution revealed that a serine cluster at the distal end of the H 4 R C-terminal tail is essential for the recruitment of β-arrestin1/2, and consequently, receptor internalization and desensitization of G protein-driven extracellular-signal-regulated kinase (ERK)1/2 phosphorylation and label-free cellular impedance. In contrast, alanine substitution of serines and threonines in the intracellular loop 3 of the H 4 R did not affect β-arrestin2 recruitment and receptor desensitization, but reduced β-arrestin1 recruitment and internalization. Hence, β-arrestin recruitment to H 4 R requires the putative phosphorylated serine cluster in the H 4 R C-terminal tail, whereas putative phosphosites in the intracellular loop 3 have different effects on β-arrestin1 versus β-arrestin2. Mutation of these putative phosphosites in either intracellular loop 3 or the C-terminal tail did not affect the histamine-induced recruitment of GRK2 and GRK3 but does change the interaction of H 4 R with GRK5 and GRK6, respectively. Identification of H 4 R interactions with these proteins is a first step in the understanding how this receptor might be dysregulated in pathophysiological conditions., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Despite the high diversity of histamine H 3 receptor (H 3 R) antagonist/inverse agonist structures, partial or full H 3 R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-yl)pyrimidin-2-amine 11b as a partial H 3 R agonist. Here, the design, synthesis, and structure-activity relationships of 11b analogues are described. This series yields several non-imidazole full agonists with potencies varying with the alkyl substitution pattern on the basic amine following the in vitro evaluation of H 3 R agonism using a cyclic adenosine monophosphate response element-luciferase reporter gene assay. The key compound VUF16839 ( 14d ) combines nanomolar on-target activity (p K i = 8.5, pEC 50 = 9.5) with weak activity on cytochrome P450 enzymes and good metabolic stability. The proposed H 3 R binding mode of 14d indicates key interactions similar to those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice revealed an amnesic effect at 5 mg/kg intraperitoneally. The excellent in vitro and in vivo pharmacological profiles and the non-imidazole structure of 14d make it a promising tool compound in H 3 R research.

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H 3 receptor (H 3 R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H 3 R ligand 44 . It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H 3 R was validated with washout experiments and leads to inverse agonism on H 3 R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H 3 R conformation and to study the consequences of prolonged inhibition of the H 3 R., Competing Interests: The authors declare no conflicts of interest.

We report a detailed structure-activity relationship for the scaffold of VUF16216, a compound we have previously communicated as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho -position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para -position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compounds. Tool compounds VUF15888 ( 4d ) and VUF16620 ( 6e ) represent more subtle efficacy switches, while VUF16216 ( 6f ) displays the largest efficacy switch, from antagonism to full agonism. The compound class disclosed here can aid in new photopharmacology studies of CXCR3 signaling., (Copyright © 2019, Gómez-Santacana et al.; licensee Beilstein-Institut.)

Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the 'effective' target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβ 1 γ 2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gα i -coupled histamine H 3 receptor (H 3 R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H 3 R responsiveness and re-equilibration of pre-bound antagonists between the tested H 3 R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix ® ) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H 3 R.

Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship between ligand structures and their binding kinetics is often poorly understood. We show that both rupatadine ( 1 ) and desloratadine ( 2 ) have a long residence time at the histamine H 1 receptor (H 1 R). Through development of a [ 3 H]levocetirizine radiolabel, we find that the residence time of 1 exceeds that of 2 more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine analogues. Methylene-linked cycloaliphatic or β-branched substitutions of desloratadine increase the residence time at the H 1 R, conveying a longer duration of receptor antagonism. However, cycloaliphatic substituents directly attached to the piperidine amine (i.e., lacking the spacer) have decreased binding affinity and residence time compared to their methylene-linked structural analogues. Guided by docking studies, steric constraints within the binding pocket are hypothesized to explain the observed differences in affinity and binding kinetics between analogues.

Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H 1 receptor (H 1 R) antagonists were determined using radioligands with either slow (low k off ) or fast (high k off ) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.

Spatiotemporal control over biochemical signaling processes involving G protein-coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H 3 receptor (hH 3 R). Upon illumination, key compound 65 decreases its affinity for the hH 3 R by 8.5-fold and its potency in hH 3 R-mediated G i protein activation by over 20-fold, with the trans and cis isomer both acting as full agonist. In real-time two-electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light-induced modulation of hH 3 R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Receptor-binding affinity and ligand-receptor residence time are key parameters for the selection of drug candidates and are routinely determined using radioligand competition-binding assays. Recently, a novel bioluminescence resonance energy transfer (BRET) method utilizing a NanoLuc-fused receptor was introduced to detect fluorescent ligand binding. Moreover, this NanoBRET method gives the opportunity to follow fluorescent ligand binding on intact cells in real time, and therefore, results might better reflect in vivo conditions as compared with the routinely used cell homogenates or purified membrane fractions. In this study, a real-time NanoBRET-based binding assay was established and validated to detect binding of unlabeled ligands to the histamine H 3 receptor (H 3 R) and histamine H 4 receptor on intact cells. Obtained residence times of clinically tested H 3 R antagonists were reflected by their duration of H 3 R antagonism in a functional receptor recovery assay., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug-target binding kinetics has recently attracted considerable interest in view of the potential predictive power for in vivo drug efficacy. The recently introduced antihistamine bilastine has a long duration of in vivo drug action, which outlasts pharmacological active bilastine concentrations in blood. To provide a molecular basis for the long duration of action, we explored the kinetics of bilastine binding to the human histamine H 1 receptor using [ 3 H]mepyramine binding studies and compared its pharmacodynamics properties to the reference compounds fexofenadine and diphenhydramine, which have a long (60 ± 20 min) and short (0.41 ± 0.1 min) residence time, respectively. Bilastine shows a long drug-target residence time at the H 1 receptor (73 ± 5 min) and this results in a prolonged H 1 receptor antagonism in vitro (Ca 2+ mobilization in Fluo-4 loaded HeLa cells), following a washout of unbound antagonist. Hence, the long residence time of bilastine can explain the observed long duration of drug action in vivo., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

For optical control of GPCR function, we set out to develop small-molecule ligands with photoswitchable efficacy in which both configurations bind the target protein but exert distinct pharmacological effects, that is, stimulate or antagonize GPCR activation. Our design was based on a previously identified efficacy hotspot for the peptidergic chemokine receptor CXCR3 and resulted in the synthesis and characterization of five new azobenzene-containing CXCR3 ligands. G protein activation assays and real-time electrophysiology experiments demonstrated photoswitching from antagonism to partial agonism and even to full agonism (compound VUF16216). SAR evaluation suggests that the size and electron-donating properties of the substituents on the inner aromatic ring are important for the efficacy photoswitching. These compounds are the first GPCR azo ligands with a nearly full efficacy photoswitch and may become valuable pharmacological tools for the optical control of peptidergic GPCR signaling., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H 3 receptor (H 3 R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H 3 R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/ cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H 3 R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H 3 R signaling.