In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z’ factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

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Currently employed histamine H2 receptor (H2R) radioligands possess several drawbacks, for example, high non-specificity, insurmountable binding, or short half-life. We report the synthesis and the chemical and pharmacological characterization of the highly stable carbamoylguanidine-type radioligand [3H]­UR-KAT479 ([3H]23), a subtype selective histamine H2 receptor G protein-biased agonist. [3H]23 was characterized by saturation, kinetic, and competition binding assays at the human, guinea pig, and mouse H2 receptors (co-)­expressed in HEK293­(T) cells. [3H]23 reversibly bound to the respective H2Rs with moderate to high affinity (human/guinea pig/mouse K d: 24/28/94 nM). In order to investigate the applicability of carbamoylguanidine-type ligands in animal studies elucidating the role of the H2R in the brain, we performed a preliminary partitioning experiment in the whole human/mouse blood, which indicated a low binding of [3H]23 to red blood cells. These properties turn [3H]23 into a powerful tool for the determination of binding affinities and demonstrate the promising pharmacokinetic profile of carbamoylguanidine-type ligands.

In an integrative approach, we studied cardiac effects of recently published novel H 2 receptor agonists in the heart of mice that overexpress the human H 2 receptor (H 2 -TG), littermate wild type control mice (WT) and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H 2 receptor agonists UR-Po563, UR-MB-158 and UR-MB-159 increased force of contraction in left atrium from H 2 -TG with pEC 50 values of 8.27, 9.38, and 8.28, respectively, but not in WT. Likewise, UR-Po563, UR-MB-158 and UR-MB-159increased the beating rate in right atrium from H 2 -TG with pEC 50 values of 9.01, 9.24, and 7.91, respectively, but not in WT. These effects could be antagonized by famotidine, a H 2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff perfused hearts from H 2 -TG but not WT. Similarly, UR-Po563, UR-MB-158 or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H 2 -TG. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H 2 -TG were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H 2 receptors in vitro and in vivo.We speculate that these compounds might be of some merit to treat neurological disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients.