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Histamine H4 receptor (H4R) orthologues are G‐protein‐coupled receptors (GPCRs) that exhibit species‐dependent basal activity. In contrast to the basally inactive mouse H4R (mH4R), human H4R (hH4R) shows a high degree of basal activity. We have performed long‐timescale molecular dynamics simulations and rigidity analyses on wild‐type hH4R, the experimentally characterized hH4R variants S179M, F169V, F169V+S179M, F168A, and on mH4R to investigate the molecular nature of the differential basal activity. H4R variant‐dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F16945.55 dips into the orthosteric binding pocket only in the case of hH4R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H4R activation that are of importance for other GPCRs. [ABSTRACT FROM AUTHOR]

New classes of alkylated hetarylpropylguanidines with different functionality and variation in spacer length were synthesized to determine their behavior at the four histamine receptor (H1R, H2R, H3R, H4R) subtypes. Alkylated guanidines with different terminal functional groups and varied basicity, like amine, guanidine and urea were developed, based on the lead structure SK&F 91486 (2). Furthermore, heteroatomic exchange at the guanidine structure of 2 led to simple analogues of the lead compound. Radioassays at all histamine receptor subtypes were accomplished, as well as organ bath studies at the guinea pig (gp) ileum (gpH1R) and right atrium (gpH2R). Ligands with terminal functionalization led to, partially, highly affine and potent structures (two digit nanomolar), which showed up a bad selectivity profile within the histamine receptor family. While the benzoylurea derivative 144 demonstrated a preference towards the human (h) H3R, S‐methylisothiourea analogue 143 obtained high affinity at the hH4R (pKi=8.14) with moderate selectivity. The molecular basis of the latter finding was supported by computational studies. Selectivity first! Structure‐activity relationship of 27 new ligands from the hetarylpropylguanidine‐type was investigated at the four histamine receptor subtypes with detailed pharmacological characterization. Methylisothiourea analogue 143 turned out to have high affinity at the hH4R with perceptible selectivity. Computational studies for 143 at both hH4R and hH3R were accomplished in order to gain more insight into the respective binding modes. [ABSTRACT FROM AUTHOR]

New classes of alkylated hetarylpropylguanidines with different functionality and variation in spacer length were synthesized to determine their behavior at the four histamine receptor (H1R, H2R, H3R, H4R) subtypes. Alkylated guanidines with different terminal functional groups and varied basicity, like amine, guanidine and urea were developed, based on the lead structure SK&F 91486 (2). Furthermore, heteroatomic exchange at the guanidine structure of 2 led to simple analogues of the lead compound. Radioassays at all histamine receptor subtypes were accomplished, as well as organ bath studies at the guinea pig (gp) ileum (gpH1R) and right atrium (gpH2R). Ligands with terminal functionalization led to, partially, highly affine and potent structures (two digit nanomolar), which showed up a bad selectivity profile within the histamine receptor family. While the benzoylurea derivative 144 demonstrated a preference towards the human (h) H3R, S‐methylisothiourea analogue 143 obtained high affinity at the hH4R (pKi=8.14) with moderate selectivity. The molecular basis of the latter finding was supported by computational studies. Selectivity first! Structure‐activity relationship of 27 new ligands from the hetarylpropylguanidine‐type was investigated at the four histamine receptor subtypes with detailed pharmacological characterization. Methylisothiourea analogue 143 turned out to have high affinity at the hH4R with perceptible selectivity. Computational studies for 143 at both hH4R and hH3R were accomplished in order to gain more insight into the respective binding modes. [ABSTRACT FROM AUTHOR]

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology [1], [2]. The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity [3]. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) [4]. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity [4], tumour [1] and bone loss [5]. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability [6]. Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors. Crystal structures of the neuropeptide Y1 receptor in complex with two distinct antagonists combined with NMR, molecular docking and mutagenesis studies inform a proposed model for receptor-agonist binding. [ABSTRACT FROM AUTHOR]

In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R. [ABSTRACT FROM AUTHOR]

The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological tools. Such differences were extremely pronounced in case of proximal readouts, e. g. [32P]GTPase or [35S]GTPγS binding assays. To improve the predictability of in vitro investigations, the aim of this study was to establish a reporter gene assay for human, murine and rat H4Rs, using bioluminescence as a more distal readout. For this purpose a cAMP responsive element (CRE) controlled luciferase reporter gene assay was established in HEK293T cells, stably expressing the human (h), the mouse (m) or the rat (r) H4R. The potencies and efficacies of 23 selected ligands (agonists, inverse agonists and antagonists) were determined and compared with the results obtained from proximal readouts. The potencies of the examined ligands at the human H4R were consistent with reported data from [32P]GTPase or [35S]GTPγS binding assays, despite a tendency toward increased intrinsic efficacies of partial agonists. The differences in potencies of individual agonists at the three H4R orthologs were generally less pronounced compared to more proximal readouts. In conclusion, the established reporter gene assay is highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [32P]GTPase and [35S]GTPγS binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways. [ABSTRACT FROM AUTHOR]

The family of neuropeptide Y (NPY) receptors comprises four subtypes (Y 1 R, Y 2 R, Y 4 R, Y 5 R), which are addressed by at least three endogenous peptides, i.e., NPY, peptide YY, and pancreatic polypeptide (PP), the latter showing a preference for Y 4 R. A series of cyclic oligopeptidic Y 4 R ligands were prepared by applying a novel approach, i.e., N-terminus to arginine side-chain cyclization. Most peptides acted as Y 4 R partial agonists, showing up to 60-fold higher Y 4 R affinity compared to the linear precursor peptides. Two cyclic hexapeptides ( 18 , 24 ) showed higher Y 4 R potency (Ca 2+ aequorin assay) and, with p K i values >10, also higher Y 4 R affinity compared to human pancreatic polypeptide (hPP). Compounds such as 18 and 24 , exhibiting considerably lower molecular weight and considerably more pronounced Y 4 R selectivity than PP and previously described dimeric peptidic ligands with high Y 4 R affinity, represent promising leads for the preparation of labeled tool compounds and might support the development of drug-like Y 4 R ligands.

Within the family of neuropeptide Y (NPY) receptors, the Y 4 receptor (Y 4 R) is unique as it prefers pancreatic polypeptide over NPY and peptide YY. Today, low-molecular-weight Y 4 R ligands are lacking, in particular antagonists. We synthesized a series of peptidic NPY Y 4 R ligands, derived from the hexapeptide acetyl-Arg-Tyr-Arg-Leu-Arg-Tyr-NH 2 ( 1 ), reported to be a Y 4 R partial agonist with high affinity (p K i Y 4 R: 8.43). Peptide 1 was N-terminally extended as well as truncated and subjected to a d-amino acid scan, and Leu was replaced by different amino acids. Compounds were characterized by radioligand competition binding and functional studies (Ca i 2+ mobilization and β-arrestin 1/2 recruitment). N-terminal truncation of 1 resulted in a tetrapeptide (Arg-Leu-Arg-Tyr-NH 2 ), being a Y 4 R partial agonist with unchanged Y 4 R affinity (p K i : 8.47). Remarkably, replacement of Leu in 1 and in derivatives of 1 by Trp turned Y 4 R agonism to antagonism, giving Y 4 R antagonists with p K i values ≤7.57.

Comprehensively characterized fluorescent probes for the histamine H 3 receptor (H 3 R) and especially for the H 4 R orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementary tools to radioligands. In view of fluorescent probes for BRET-based binding studies and for localizing the H 4 R in live cells, we synthesized and biologically characterized Py-5-labeled histamine derivatives. The most notable compound was UR-DEBa242 ( 26 , 1-[4-(1 H -Imidazol-4-yl)butyl]-4-{(1 E ,3 E )-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}-2,6-dimethylpyridinium hydrotrifluoroacetate trifluoroacetate), acting as a partial agonist at the hH 3 R [pEC 50 (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mH 4 Rs [pIC 50 (reporter gene) 8.76/7.08; pIC 50 /p K b (β-arrestin2) 7.81/7.30]. In confocal microscopy, 26 proved suitable for hH 4 R localization and trafficking studies in live cells. BRET-based binding at the NLuc-hH 3,4 Rs/mH 4 R [p K d 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (∼2 min)] revealed 26 as a useful molecular tool to determine hH 3,4 Rs/mH 4 R binding affinities of ligands binding to these receptors.

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M 2 R affinities (p K i (radioligand competition binding): 9.10-9.59). Binding studies at M 1 and M 3 -M 5 receptors indicated a M 2 R preference. Flow cytometric and high-content imaging saturation and competition binding (M 1 R, M 2 R, and M 4 R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM 2 R cells). Results from dissociation and saturation binding experiments (M 2 R) in the presence of allosteric M 2 R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M 2 R in imaging and binding studies and suggest that they have a dualsteric mode of action.

The recently resolved crystal structure of the neuropeptide Y Y 1 receptor (Y 1 R), co-crystallized with the high-affinity (p K i : 10.11), argininamide-type Y 1 R antagonist UR-MK299 ( 2 ), revealed that the N ω -carbamoyl substituent (van der Waals volume: 139 Å 3 ) is deeply buried in the receptor, occupying a hydrophobic pocket. We synthesized and characterized a series of argininamides, structurally related to 2 . Y 1 R affinity decreased with increasing size of the carbamoyl residue (minimal p K i : 5.67). Exceeding a critical size of the substituent (van der Waals volume: 212 Å 3 ), the ligands bound in an inverted mode with the carbamoyl side chain located at the surface of the receptor, as suggested by induced-fit docking and MD simulations., (This journal is © The Royal Society of Chemistry 2020.)

Histamine H 4 receptor (H 4 R) orthologues are G-protein-coupled receptors (GPCRs) that exhibit species-dependent basal activity. In contrast to the basally inactive mouse H 4 R (mH 4 R), human H 4 R (hH 4 R) shows a high degree of basal activity. We have performed long-timescale molecular dynamics simulations and rigidity analyses on wild-type hH 4 R, the experimentally characterized hH 4 R variants S179M, F169V, F169V+S179M, F168A, and on mH 4 R to investigate the molecular nature of the differential basal activity. H 4 R variant-dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F169 45.55 dips into the orthosteric binding pocket only in the case of hH 4 R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H 4 R activation that are of importance for other GPCRs., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Muscarinic acetylcholine receptors (MRs), comprising five subtypes (M 1 R-M 5 R) in humans, exhibit a high degree of structural similarity. Therefore, subtype-selective MR agonists and antagonists are lacking. We present an approach to highly M 2 R-selective MR antagonists based on the conjugation of di- or tripeptides to M 2 R-preferring dibenzodiazepinone-type MR antagonists. M 2 R selectivity was dependent on the peptide sequence and on the type of linker. The introduction of basic amino acids resulted in improved M 2 R selectivity (e.g., UR-AP148 (48): p K i (hM 2 R) of 8.97, ratio of K i M 1 R/M 2 R/M 3 R/M 4 R/M 5 R of 49:1:6500:60:400) compared to reported pyridobenzo- and dibenzodiazepinone-type MR ligands. A supposed dualsteric binding mode of the DIBA-peptide conjugates, such as 48, at MRs was supported by molecular dynamics simulations.

The dualsteric ligand approach, aiming at ligands with improved subtype selectivity, has been increasingly applied to muscarinic receptors (MRs). In this article, we present the synthesis and characterization of a M 2 R subtype-preferring radiolabeled dibenzodiazepinone-type antagonist ([ 3 H]UNSW-MK259, [ 3 H]19) and its homodimeric analogue [ 3 H]UR-AP060 ([ 3 H]33). Saturation binding studies at the M 2 R, using the orthosteric antagonist atropine to determine unspecific binding, proved that the monomeric and the dimeric compound bind to the orthosteric binding site (apparent K d : 0.87 and 0.31 nM, respectively). Various binding studies with [ 3 H]19 and [ 3 H]33 at the M 2 R, for instance, saturation binding experiments in the presence of the allosteric MR modulators W84 (8) or LY2119620 (9) (Schild-like analysis) suggested a competitive mechanism between the allosteric modulator and the dibenzodiazepinone derivatives, and thus a dualsteric binding mode of both 19 and 33. This was consistent with the results of M 2 R MD simulations (≥2 μs) performed with 19 and 33.