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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.)

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.)

Heteromerization of G protein-coupled receptors (GPCRs) can significantly change the functional properties of involved receptors. Various biochemical and biophysical methodologies have been developed in the last two decades to identify and functionally evaluate GPCR heteromers in heterologous cells, with recent approaches focusing on GPCR complex stoichiometry and stability. Yet validation of these observations in native tissues is still lagging behind for the majority of GPCR heteromers. Remarkably, recent studies, particularly some involving advanced fluorescence microscopy techniques, are contributing to our current knowledge of aspects that were not well known until now, such as GPCR complex stoichiometry and stability. In parallel, a growing effort is being applied to move the field forward into native systems. This short review will highlight recent developments to study the stoichiometry and stability of GPCR complexes and methodologies to detect native GPCR dimers., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein-coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

After the recent description of β-arrestin2 recruitment to the human histamine H₄ receptor (hH₄R) in response to the well known H₄R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120), we evaluated in this study the efficacy of 31 known hH₄R ligands to induce Gα(i) protein signaling and β-arrestin2 recruitment by the hH₄R. The selected hH(4)R ligands belong to nine different structural classes that partly cover (pre)clinical trial candidates. We have identified hH₄R ligands with a significant bias for the Gα(i) protein or β-arrestin2 pathway on the basis of efficacy differences. In addition, hH₄R antagonists that did not show positive efficacy in either functional readouts were found. A common trend in pathway preference for the nine different ligand classes could not be observed. In particular, the isothiourea class shows very diverse results, varying from Gα(i) protein-biased or β-arrestin2-biased to nonbiased antagonists upon minor structural changes. The identified biased hH₄R ligands are important pharmacological tools to unravel the significance of biased hH₄R signaling in H₄R pharmacology.

Human herpesviruses (HHVs) are widespread pathogens involved in proliferative diseases, inflammatory conditions, and cardiovascular diseases. During evolution, homologs of human chemokine receptors were integrated into the HHV genomes. In addition to binding endogenous chemokines, these viral G protein-coupled receptors (vGPCRs) have acquired the ability to signal in a constitutive manner. Ligand-induced and ligand-independent and autocrine and paracrine signaling properties of vGPCRs modify the functions of the expressing cells and lead to transformation and escape from immune surveillance. Furthermore, cross-talk or heterodimerization with endogenous chemokine receptors represent other ways for vGPCRs to modify intracellular signaling and cellular functions. As such, these viral receptors seem to play a prominent role during viral pathogenesis and life cycle and thus represent innovative antiviral therapies.

The human herpes virus 6 (HHV-6)-encoded chemokine receptor U51 constitutively activates phospholipase C (PLC) and inhibits cAMP-responsive element (CRE)-mediated gene transcription via the activation of G(q/11) proteins. Yet, chemokines known to bind U51 differentially regulate U51 coupling to G proteins. CCL5/RANTES induced pertussis toxin (PTX)-insensitive increases in PLC activity and changes in intracellular free calcium concentration ([Ca2+]i), whereas both CCL2/MCP-1 and CCL11/eotaxin failed to stimulate PLC activity or increase [Ca2+]i. In contrast, all three chemokines counteracted the effects of U51 on CRE activity via the activation of PTX-sensitive G(i/o) proteins. For each of the tested chemokines, coexpression of U51 with a variety of G alpha subunits, however, revealed a distinct profile for preferred G-protein coupling, which could be shifted by modulation of the relative expression of G proteins. These findings are consistent with a chemokine-selective trafficking of receptor stimulus to distinct G proteins and suggest that the constitutive activity of U51 and the chemokine-induced signaling involve different active states of the receptor. By virtue of its ability to constitutively activate signaling pathways, its G-protein promiscuity, and the chemokine-directed trafficking of receptor stimulus, U51 can be considered a sensitive and versatile virally encoded signaling device, potentially of importance in HHV-6-related pathologies.