Radioligands used previously for histamine H3 receptor (H3R) are accompanied by a number of disadvantages. In this study, we report the synthesis of the new H3R radioligand [3H]UR-MN259 ([3H]11) with high (radio)chemical purity and stability. The radioligand exhibits sub-nanomolar affinity for the target receptor (pKi (H3R) = 9.56) and displays an outstanding selectivity profile within the histamine receptor family (>100,000-fold selective). [3H]UR-MN259 is ideally suitable for the characterization of H3R ligands in competition binding and shows one-site binding to the H3R in saturation binding experiments. The radiotracer shows fast association to the receptor (τassoc = 6.11 min), as well as full dissociation from the receptor (τdissoc = 14.48 min) in kinetic binding studies. The distinguished profile of [3H]UR-MN259 makes it a highly promising pharmacological tool to further investigate the role of the H3R in the CNS.

In the search for potential bioisosteres of the 4-imidazolyl ring in acylguanidines (e. g. UR-AK24), known to possess affinity to several histamine receptor subtypes (HxR, x = 1-4), and cyanoguanidine- type H4R agonists (e. g. UR-PI376), the contribution of various heterocycles to agonism, antagonism and HR subtype selectivity was studied (recombinant human H(1,2,3,4)Rs, isolated guinea pig organs (H1R, H2R)). While minor structural modifications of UR- PI376 analogues were not tolerated regarding H4R agonism, in the case of the acylguanidines, a 1,2,4-triazole ring shifted the selectivity toward the H2R.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The four functionally expressed human neuropeptide Y receptor subtypes (hY1R, hY2R, hY4R, hY5R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive Gi/o-proteins. The number of small molecules described as ligands for hY1R and hY5R exceeds by far those for hY2R. Potent non-peptidergic ligands for the hY4R are not available so far. Here, we report on the functional reconstitution of the hY2R and the hY4R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the Gi/o family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y2) and hPP (Y4), the GPCRs coupled to various Gi/Go-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY2R + Gαi2 and hY4R + Gαi2/Gαo + RGS4, combined with Gβ1γ2, yielded best signal-to-noise ratios. hY2R function was validated using both agonistic peptides (NPY, PYY, NPY13–36) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY4R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY2R and abolished hY4R function. Investigations with monovalent salts showed sensitivity of hY4R toward Na+, revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y4R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y2 and Y4 receptor ligands.

The histamine H 4 receptor (H 4 R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H 4 R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H 4 R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H 4 R in comparison to human H 4 R expressed in Sf9 insect cells. Only hH 4 R and cH 4 R exhibited a sufficiently high [ 3 H]histamine affinity for radioligand binding studies. Generally, cH 4 R exhibited lower ligand-affinities than hH 4 R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH 4 R than at other H 4 R species orthologs. Unlike the other H 4 R species orthologs, hH 4 R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H 4 R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH 4 R, rH 4 R, and mH 4 R, whereas at hH 4 R, JNJ7777120 was a partial inverse agonist. H 4 R agonists from the class of N G -acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H 4 R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H 4 R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H 4 R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of “the prototypical H 4 R antagonist” JNJ7777120 as H 4 R antagonism.

Guanidine and acylguanidine groups are crucial structural features of numerous biologically active compounds. Depending on the biological target, acylguanidines may be considered as considerably less basic bioisosteres of guanidines with improved pharmacokinetics and pharmacodynamics, as recently reported for N'-monoalkylated N-acylguanidines as ligands of G-protein-coupled receptors (GPCRs). The molecular basis for enhanced ligand-receptor interactions of acylguanidines is far from being understood. So far, only a few and contradictory results about their conformational preferences have been reported. In this study, the conformations, conformational preferences, and conformational exchange of four unprotonated and seven protonated monoalkylated acylguanidines with up to six anions and with bisphosphonate tweezers are investigated by NMR. Furthermore, the effects of the acceptor properties in acylguanidine salts, of microsolvation by dimethylsulfoxide, and of varying acyl and alkyl substituents are studied. Throughout the whole study, exclusively two out of eight possible acylguanidine conformations were detected, independent of the compound, the anion, or the solvent used. For the first time, it is shown that the strength and number of intermolecular interactions with anions, solvent molecules, or biomimetic receptors decide the conformational preferences and exchange rates. One recently presented and two new crystal structures resemble the conformational preferences observed in solution. Thus, consistent conformational trends are found throughout the structurally diverse compound pool, including two potent GPCR ligands, different anions, and receptors. The presented results may contribute to a better understanding of the mechanism of action at the molecular level and to the prediction and rational design of these biologically active compounds.

Since its discovery 10 years ago the histamine H(4) receptor (H(4)R) has attracted attention as a potential drug target, for instance, for the treatment of inflammatory and allergic diseases. Potent and selective ligands including agonists are required as pharmacological tools to study the role of the H(4)R in vitro and in vivo. Many H(4)R agonists, which were identified among already known histamine receptor ligands, show only low or insufficient H(4)R selectivity. In addition, the investigation of numerous H(4)R agonists in animal models is hampered by species-dependent discrepancies regarding potencies and histamine receptor selectivities of the available compounds, especially when comparing human and rodent receptors. This article gives an overview about structures, potencies, and selectivities of various compounds showing H(4)R agonistic activity and summarizes the structure-activity relationships of selected compound classes.

3-(1H-Imidazol-4-yl)propylguanidine (SK&F 91486, 4) was identified as a potent partial agonist at the human histamine H(3) receptor (hH(3)R) and human histamine H(4) receptor (hH(4)R). With the aim to increase selectivity for the hH(4)R, the guanidine group in 4 was acylated. N(1)-Acetyl-N(2)-[3-(1H-imidazol-4-yl)propyl]guanidine (UR-PI288, 13) was a potent full agonist at the hH(4)R (pEC(50) = 8.31; alpha = 1.00), possessing more than 1000- and 100-fold selectivity relative to the hH(1)R and hH(2)R, respectively, and possessing only low intrinsic activity (alpha = 0.27) at the hH(3)R.

This study reports the synthesis and pharmacological characterization of tritium-labeled N(1)-[3-(1H-imidazol-4-yl)propyl]-N(2)-propionylguanidine ([(3)H]UR-PI294), a novel and readily accessible radioligand for the human histamine H(3) receptor (hH(3)R) and H(4) receptor (hH(4)R). The radioligand displays high affinity for both histamine receptor subtypes (K(D) (hH(3)R)=1.1 nM, K(D) (hH(4)R)=5.1 nM) and is shown to be a valuable pharmacological tool for the determination of hH(3)R and hH(4)R affinities.

Recently, we identified high-affinity human histamine H-3 (hH(3)R) and H-4 receptor (hH(4)R) ligands among a series of N-G-acylated imidazolylpropylguanidines, which were originally designed as histamine H-2 receptor (H2R) agonists. Aiming at selectivity for hH(4)R, the acylguanidine group was replaced with related moieties. Within a series of cyanoguanidines, 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine (UR-PI376, 67) was identified as the most potent hH(4)R agonist (pEC(50) = 7.47, alpha = 0.93) showing negligible hH(1)R and hH(2)R activities and significant selectivity over the hH(3)R (pK(B) = 6.00, alpha = -0.28), as determined in steady-state GTPase assays using. membrane preparations of hH(x)R-expressing Sf9 cells. In contrast to previously described selective H4R agonists, this compound and other 3-substituted derivatives are devoid of agonistic activity at the other HR subtypes. Modeling of the binding mode of 67 suggests that the cyanoguanidine moiety forms charge-assisted hydrogen bonds not only with the conserved Asp-94 but also with the hH(4)R-specific Arg-341 residue. 2-Carbamoyl-1-[2-(1H-imidazol-4-yl)ethyl]-3-(3-phenylpropyl)guanidine (UR-PI97, 88) was unexpectedly identified as a highly potent and selective hH(3)R inverse agonist (pK(B) = 8.42, > 300-fold selectivity over the other HR subtypes).

N-1-Aryl(heteroaryl)alkyl-N-2-[3-(1H-imidazol-4-yl)propyl]guanidines are potent histamine H-2-receptor (H2R) agonists, but their applicability is compromised by the lack of oral bioavailability and CNS penetration. To improve pharmacokinetics, we introduced carbonyl instead of methylene adjacent to the guanidine moiety, decreasing the basicity of the novel H2R agonists by 4-5 orders of magnitude. Some acylguanidines with one phenyl ring were even more potent than their diaryl analogues. As demonstrated by HPLC-MS, the acylguanidines (bioisosteres of the alkylguanidines) were absorbed from the gut of mice and detected in brain. In GTPase assays using recombinant receptors, acylguanidines were more potent at the guinea pig than at the human H2R. At the hH(1)R and hH(3)R, the compounds were weak to moderate antagonists or partial agonists. Moreover, potent partial hH(4)R agonists were identified. Receptor subtype selectivity depends on the imidazolylpropylguanidine moiety (privileged structure), opening an avenue to distinct pharmacological tools including potent H4R agonists.