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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 H3 receptor (H3R) and histamine H4 receptor on intact cells. Obtained residence times of clinically tested H3R antagonists were reflected by their duration of H3R antagonism in a functional receptor recovery assay.

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

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 Gαi protein signaling and β-arrestin2 recruitment by the hH4R. The selected hH4R 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 Gαi 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 Gαi 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.

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.

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.