Probing the Histamine H Receptor Binding Site to Explore Ligand Binding Kinetics.

Analysis of structure-kinetic relationships (SKR) can contribute to an improved understanding of receptor-ligand interactions. Here, fragment (4-(2-benzylphenoxy)-1-methylpiperidine) was used in different fragment growing approaches to mimic the putative binding mode of the long residence time (RT) ligands olopatadine, acrivastine, and levocetirizine at the histamine H receptor (HR). SKR analyses reveal that introduction of a carboxylic acid moiety can increase RT at HR up to 11-fold.

Histamine H Receptor Isoforms: Insights from Alternative Splicing to Functional Complexity.

Alternative splicing significantly enhances the diversity of the G protein-coupled receptor (GPCR) family, including the histamine H receptor (HR). This post-transcriptional modification generates multiple HR isoforms with potentially distinct pharmacological and physiological profiles. HR is primarily involved in the presynaptic inhibition of neurotransmitter release in the central nervous system.

A high-affinity, cis-on photoswitchable beta blocker to optically control β-adrenergic receptors in vitro and in vivo.

This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of β-adrenoceptor (βAR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSS (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 βAR 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.

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

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.

Synthesis and Pharmacological Characterization of New Photocaged Agonists for Histamine H and H 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 H receptors (HR) and H receptors (HR).

Pharmacological characterization of seven human histamine H receptor isoforms.

The histamine H receptor (HR) 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 HR 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 protein signaling. The last two decades HR drug discovery and lead optimization has been exclusively focused on the 445 amino acids-long reference isoform HR-445.

Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

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 -arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC = 8.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

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.

SAR exploration of the non-imidazole histamine H receptor ligand ZEL-H16 reveals potent inverse agonism.

Histamine H receptor (H 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 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 R affinity.

Optical control of the β-adrenergic receptor with opto-prop-2: A -active azobenzene analog of propranolol.

In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors β-AR and β-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 - isomerization (>94%) and good thermal stability (  > 10 days) of the resulting -isomer in an aqueous buffer. Upon illumination with 360-nm light to PSS , large differences in binding affinities were observed for photoswitchable compounds at β-AR as well as β-AR.

A NanoBRET-Based HR Conformational Biosensor to Study Real-Time H 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 H receptor (HR) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with HR ligands. In the current study, we have further characterized this HR 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.

New Chemical Biology Tools for the Histamine Receptor Family.

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.

Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H Receptor.

The histamine H receptor (HR) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity HR-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a HR 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 HR fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore.

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins.

The histamine H receptor (HR) 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, HR is a well-established therapeutic target for antihistamines that relieve allergy symptoms. HR signals via heterotrimeric G proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2.

Optical control of Class A G protein-coupled receptors with photoswitchable ligands.

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).

Identification of TSPAN4 as Novel Histamine H Receptor Interactor.

The histamine H receptor (HR) 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 HR 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.

NanoLuc-Based Methods to Measure β-Arrestin2 Recruitment to G Protein-Coupled Receptors.

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 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput.

Exploring the Effect of Cyclization of Histamine H Receptor Antagonists on Ligand Binding Kinetics.

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 receptor (HR), a member of the family A G-protein coupled receptor. In this study, we show that the binding kinetics of HR antagonists at the HR is dependent on the cyclicity of both the aromatic head group and the amine moiety of HR ligands, the chemotypes that are characteristic for the first-generation HR antagonists.

Analysis of Missense Variants in the Human Histamine Receptor Family Reveals Increased Constitutive Activity of E410K Variant in the Histamine H Receptor.

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.

Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization.

: 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.

Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways.

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.

Development of a Conformational Histamine H Receptor Biosensor for the Synchronous Screening of Agonists and Inverse Agonists.

The histamine H receptor (HR) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel HR targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect HR modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular HR 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 HR 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 receptor ligands with unprecedented accuracy.

Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H Receptor in β-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling.

The histamine H receptor (HR) activates Gα-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 HR than β-arrestin1 and 2 upon agonist stimulation, whereas receptor internalization dynamics toward early endosomes was slower.