Andrea Strasser

Species-dependent activities of G-protein-coupled receptor ligands: lessons from histamine receptor orthologs

Histamine is a biogenic amine that exerts its biological effects as a neurotransmitter and local mediator via four histamine receptor (HR) subtypes (H(x)Rs) - H(1)R, H(2)R, H(3)R, and H(4)R - belonging to the superfamily of G-protein-coupled receptors (GPCRs). All four H(x)Rs exhibit pronounced differences in agonist and/or antagonist pharmacology among various species orthologs. The species differences constitute a problem for animal experiments and drug development. This problem applies to GPCRs with diverse ligands. Here, we summarize our current knowledge on H(x)R orthologs as a case study for species-dependent activity of GPCR ligands. We show that species-specific pharmacology also provides unique opportunities to study important aspects of GPCR pharmacology in general, including ligand-binding sites, the roles of extracellular domains in ligand binding and receptor activation, agonist-independent (constitutive) receptor activity, thermodynamics of ligand/receptor interaction, receptor-activation mechanisms, and ligand-specific receptor conformations.

Interactions of Histamine H1-Receptor Agonists and Antagonists with the Human Histamine H4-Receptor

The human histamine H4-receptor (hH4R) possesses high constitutive activity and, like the human H1-receptor (hH1R), is involved in the pathogenesis of type-I allergic reactions. The study aims were to explore the value of dual H1/H4R antagonists as antiallergy drugs and to address the question of whether H1R ligands bind to hH4R. In an acute murine asthma model, the H1R antagonist mepyramine and the H4R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120) exhibited synergistic inhibitory effects on eosinophil accumulation in the bronchoalveolar lavage fluid. At the hH4R expressed in Sf9 insect cells, 18 H1R antagonists and 22 H1R agonists showed lower affinity to hH4R than to hH1R as assessed in competition binding experiments. For a small number of H1R antagonists, hH4R partial agonism was observed in the steady-state GTPase assay. Most compounds were neutral antagonists or inverse agonists. Twelve phenylhistamine-type hH1R partial agonists were also hH4R partial agonists. Four histaprodifen-type hH1R partial agonists were hH4R inverse agonists. Dimeric histaprodifen was a more efficacious hH4R inverse agonist than the reference compound thioperamide. Suprahistaprodifen was the only histaprodifen acting as hH4R partial agonist. Suprahistaprodifen was docked into the binding pocket of inactive and active hH4R models in two different orientations, predominantly stabilizing the active state of hH4R. Collectively, the synergistic effects of H1R and H4R antagonists in an acute asthma model and the overlapping interaction of structurally diverse H1R ligands with hH1R and hH4R indicate that the development of dual H1R/H4R antagonists is a worthwhile and technically feasible goal for the treatment of type-I allergic reactions.

Synthesis and structure-activity relationships of cyanoguanidine-type and structurally related histamine H4 receptor agonists.

Recently, we identified high-affinity human histamine H3 (hH3R) and H4 receptor (hH4R) ligands among a series of NG-acylated imidazolylpropylguanidines, which were originally designed as histamine H2 receptor (H2R) agonists. Aiming at selectivity for hH4R, 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 hH4R agonist (pEC50 = 7.47, alpha = 0.93) showing negligible hH1R and hH2R activities and significant selectivity over the hH3R (pKB = 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 hH4R-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 hH3R inverse agonist (pKB = 8.42, >300-fold selectivity over the other HR subtypes).

Impact of the DRY Motif and the Missing “Ionic Lock” on Constitutive Activity and G-Protein Coupling of the Human Histamine H4 Receptor

It is assumed that many G protein-coupled receptors (GPCRs) are restrained in an inactive state by the “ionic lock,” an interaction between an arginine in transmembrane domain (TM) 3 (R3.50) and a negatively charged residue in TM6 (D/E6.30). In the human histamine H4 receptor (hH4R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH4R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. The role of R3.50 was investigated by generating hH4R-R3.50A. Both mutants were expressed alone or together with Gαi2 and Gβ1γ2 in Sf9 cells and characterized in GTPase, 35S-labeled guanosine 5′-[γ-thio]triphosphate binding, and high-affinity agonist binding assays. Unexpectedly, compared with hH4R, hH4R-A6.30E showed only nonsignificant reduction of constitutive activity and G protein-coupling efficiency. The KD of [3H]histamine was unaltered. By contrast, hH4R-R3.50A did not stimulate G proteins. Thioperamide affinity at hH4R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH4R state in complex with the Gαi2 C terminus was compared with the crystal structures of turkey β1 and human β2 adrenoceptors. We conclude that 1) constitutive activity of hH4R is facilitated by the salt bridge D5.69-R6.31 rather than by the missing ionic lock, 2) Y3.60 may form alternative locks in active and inactive GPCR states, 3) R3.50 is crucial for hH4R–G protein coupling, and 4) hH4R-R3.50A represents an inactive state with increased inverse agonist and reduced agonist affinity. Thus, the ionic lock, although stabilizing the inactive rhodopsin state, is not generally important for all class A GPCRs.

Pharmacological Profile of Histaprodifens at Four Recombinant Histamine H1 Receptor Species Isoforms

There are differences in the pharmacological properties of phenylhistamines and histaprodifens between guinea pig histamine H1 receptor (gpH1R) and human histamine H1 receptor (hH1R). The aim of this study was to analyze species differences in more detail, focusing on histaprodifen derivatives and including the bovine histamine H1 receptor (bH1R) and rat histamine H1 receptor (rH1R). H1R species isoforms were coexpressed with the regulator of G protein signaling RGS4 in Sf9 insect cells. We performed [3H]mepyramine binding assays and steady-state GTPase assays. For a novel class of histaprodifens, the chiral histaprodifens, unique species differences between hH1R, bH1R, rH1R, and gpH1R were observed. The chiral histaprodifens 8R and 8S were both partial agonists at gpH1R, but only 8R was a partial agonist at the other H1R species isoforms. An additional phenyl group in chiral histaprodifens 10R and 10S, respectively, resulted in a switch from agonism at gpH1Rto antagonism at hH1R, bH1R, and rH1R. In general, histaprodifens showed the order of potency hH1R < bH1R < rH1R < gpH1R. An active-state model of gpH1R was generated with molecular dynamics simulations. Dimeric histaprodifen was docked into the binding pocket of gpH1R. Hydrogen bonds and electrostatic interactions were detected between dimeric histaprodifen and Asp-116, Ser-120, Lys-187, Glu-190, and Tyr-432. We conclude the following: 1) chiral histaprodifens interact differentially with H1R species isoforms; 2) gpH1R and rH1R, on one hand, and hH1R and bH1R, on the other hand, resemble each other structurally and pharmacologically; and 3) histaprodifens interact with H1R at multiple sites.

Structural Requirements for Inverse Agonism and Neutral Antagonism of Indole-, Benzimidazole-, and Thienopyrrole-Derived Histamine H4 Receptor Ligands

The human histamine H4 receptor (hH4R), coexpressed with Gαi2 and Gβ1γ2 in Sf9 insect cells, is highly constitutively active, and thioperamide [THIO; N-cyclohexyl-4-(imidazol-4-yl)-1-piperidinecarbothioamide] is one of the most efficacious hH4R inverse agonists. High constitutive hH4R activity may have pathophysiological implications in which case inverse agonists may behave differently than neutral antagonists. To learn more about the structural requirements for hH4R inverse agonism, we investigated 25 compounds (indole, benzimidazole, and thienopyrrole derivatives) structurally related to the standard antagonist JNJ-7777120 [1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine]. We characterized the compounds in radioligand binding assays by using [3H]histamine ([3H]HA) and in steady-state GTPase assays in the presence (antagonist mode) and absence (inverse agonist mode) of the agonist HA, yielding the following results: 1) Twenty-two compounds were inverse agonists (efficacy: 15–62% of the THIO effect), and only three compounds (12%) showed neutral antagonism. Thus, inverse agonism is far more common than neutral antagonism. 2) The inverse agonistic efficacy of the R5-monosubstituted indole-derived compounds increased with the volume of R5. R5 may interact with Trp6.48 of the rotamer toggle switch and stabilize the inactive receptor conformation. 3) A subset of compounds showed large differences between the Ki value from [3H]HA competition binding and the EC50 value from steady-state GTPase assays, whereas the Kb values were closer to the Ki values. Thus, the two-state model should be extended to a model comprising a constitutively active hH4R state, which can be discriminated by inverse agonists from a structurally distinct HA-stabilized active state.

Ligand-Specific Contribution of the N Terminus and E2-Loop to Pharmacological Properties of the Histamine H1-Receptor

There are species differences between human histamine H1 receptor (hH1R) and guinea pig (gp) histamine H1 receptor (gpH1R) for phenylhistamines and histaprodifens. Several studies showed participation of the second extracellular loop (E2-loop) in ligand binding for some G protein-coupled receptors (GPCRs). Because there are large species differences in the amino acid sequence between hH1R and gpH1R for the N terminus and E2-loop, we generated chimeric hH1Rs with gp E2-loop (hgpE2H1R) and gp N terminus and gp E2-loop (hgpNgpE2H1R). hH1R, gpH1R, and chimeras were expressed in Sf9 insect cells. [3H]Mepyramine binding assays and steady-state GTPase assays were performed. In the series hH1R > hgpE2H1R > hgpNgpE2H1R, we observed a significant decrease in potency of histamine 1 in the GTPase assay. For phenoprodifen 5 and the chiral phenoprodifens 6R and 6S, a significant decrease in affinity and potency was found in the series hH1R > hgpE2H1R > hgpNgpE2H1R. In addition, we constructed new active-state H1R models based on the crystal structure of the human β2-adrenergic receptor (hβ2AR). Compared with the H1R active-state models based on the crystal structure of bovine rhodopsin, the E2-loop differs in its contact to the ligand bound in the binding pocket. In the bovine rhodopsin-based model, the backbone carbonyl of Lys187 (gpH1R) interacts with large histaprodifens in the binding pocket, but in the hβ2AR-based model, Lys187 (gpH1R) is located distantly from the binding pocket. In conclusion, the differences in N terminus and E2-loop between hH1R and gpH1R exert an influence on affinity and/or potency for histamine and phenoprodifens 5, 6R, and 6S.

Synthesis, biological evaluation, and computational studies of Tri- and tetracyclic nitrogen-bridgehead compounds as potent dual-acting AChE inhibitors and hH3 receptor antagonists.

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimers disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R.

Comparison of the pharmacological properties of human and rat histamine H3-receptors

Ligand pharmacology of histamine H(3)-receptors is species-dependent. In previous studies, two amino acids in transmembrane domain 3 (TM III) were shown to play a significant role. In this study, we characterized human and rat histamine H(3)-receptors (hH(3)R and rH(3)R, respectively), co-expressed with mammalian G proteins in Sf9 insect cell membranes. We compared a series of imidazole-containing H(3)R ligands in radioligand binding and steady-state GTPase assays. H(3)Rs similarly coupled to Gα(i/o)-proteins. Affinities and potencies of the agonists histamine, N(α)-methylhistamine and R-(α)-methylhistamine were in the same range. Imetit was only a partial agonist. The pharmacology of imetit and proxifan was similar at both species. However, impentamine was more potent and efficacious at rH(3)R. The inverse agonists ciproxifan and thioperamide showed higher potency but lower efficacy at rH(3)R. Clobenpropit was not species-selective. Strikingly, imoproxifan was almost full agonist at hH(3)R, but an inverse agonist at rH(3)R. Imoproxifan was docked into the binding pocket of inactive and active hH(3)R- and rH(3)R-models and molecular dynamic simulations were performed. Imoproxifan bound to hH(3)R and rH(3)R in E-configuration, which represents the trans-isomer of the oxime-moiety as determined in crystallization studies, and stabilized active hH(3)R-, but inactive rH(3)R-conformations. Large differences in electrostatic surfaces between TM III and TM V cause differential orientation of the oxime-moiety of imoproxifan, which then differently interacts with the rotamer toggle switch Trp(6.48) in TM VI. Collectively, the substantial species differences at H(3)Rs are explained at a molecular level by the use of novel H(3)R active-state models.

Contribution of Binding Enthalpy and Entropy to Affinity of Antagonist and Agonist Binding at Human and Guinea Pig Histamine H1-Receptor

For several GPCRs, discrimination between agonism and antagonism is possible on the basis of thermodynamics parameters, such as binding enthalpy and entropy. In this study, we analyze whether agonists and antagonists can also be discriminated thermodynamically at the histamine H1 receptor (H1R). Because previous studies revealed species differences in pharmacology between human H1R (hH1R) and guinea pig H1R (gpH1R), we analyzed a broad spectrum of H1R antagonists and agonists at hH1R and gpH1R. [3H]Mepyramine competition binding assay were performed at five different temperatures in a range from 283.15 to 303.15 K. In addition, we performed a temperature-dependent three-dimensional quantitative structure activity relationship study to predict binding enthalpy and entropy for histaprodifen derivatives, which can bind to H1R in two different orientations. Our studies revealed significant species differences in binding enthalpy and entropy between hH1R and gpH1R for some antagonists and agonists. Furthermore, in some cases, we found changes in heat capacity of the binding process that were different from zero. Differences in flexibility of the ligands may be responsible for this observation. For most ligands, the binding process to hH1R and gpH1R is clearly entropy-driven. In contrast, for the endogenous ligand histamine, the binding process is significantly enthalpy-driven at both species isoforms. Thus, a definite discrimination between antagonism and agonism based on thermodynamic parameters is possible for neither hH1R nor gpH1R, but thermodynamic analysis of ligand-binding may be a novel approach to dissect agonist- and antagonist-specific receptor conformations.