Hans-Joachim Wittmann

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.

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.

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.

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.

Molecular basis for the selective interaction of synthetic agonists with the human histamine H1-receptor compared with the guinea pig H1-receptor.

Previous studies revealed that phenylhistamines and histaprodifens possess higher potency and affinity at guinea pig histamine H1-receptor (gpH1R) than at human histamine H1-receptor (hH1R). However, we recently identified an imidazolylpropylguanidine [N1-(3-cyclohexylbutanoyl)-N2-[3-(1H-imidazol-4-yl)-propyl]guanidine (UR-AK57)] with higher potency and efficacy at hH1R compared with gpH1R. The aim of this study was to reveal the molecular basis for the species differences of synthetic ligands. We studied 11 novel phenylhistamines and phenoprodifens. H1R species isoforms were expressed in Sf9 insect cells, and [3H]mepyramine competition binding and GTPase assays were performed. We identified bulky phenylhistamines with higher potency and affinity at hH1R compared with gpH1R. Molecular dynamics simulations of ligand-H1R interactions revealed four potential binding modes for phenylhistamines possessing an additional histamine moiety; the terminal histamine moiety showed a high flexibility in the binding pocket. There are striking similarities in ligand properties in bulky phenylhistamines and UR-AK57. Comparison of bulky phenylhistamine binding mode with binding mode of UR-AK57 suggests that only one of these four binding modes should be established. The higher potency is explained by more effective van der Waals interaction of the compounds with Asn2.61 (hH1R) relative to Ser2.61 (gpH1R). In addition, two stable binding modes for phenoprodifens with different orientations in the binding-pocket were identified. Depending on phenoprodifen orientation, the highly conserved Trp6.48, part of the toggle switch involved in receptor activation, was found in an inactive or active conformation, respectively. We identified the first phenylhistamines with higher potency at hH1R than at gpH1R and obtained insight into the binding mode of bulky phenylhistamines and imidazolylpropylguanidines.

New insights into the cross-linking and degradation mechanism of Diels–Alder hydrogels

Eight-armed poly(ethylene glycol) was functionalized with furyl and maleimide groups. The two macromonomers were cross-linked by Diels-Alder (DA) reactions and the degradation behavior of the formed hydrogels was investigated. UV spectroscopy showed that maleimide groups were subject to ring-opening hydrolysis above pH 5.5, with the reaction rate depending on the pH and temperature. As a result of this, the gelation kinetics and stiffness of DA hydrogels were dependent on the temperature and the pH of the cross-linking medium, as demonstrated by rheological experiments. The gel time varied between 87.8 min (pH 3.0, 37 °C) and 374.7 min (pH 7.4, 20 °C). Values between 420 Pa (pH 9.0, 37 °C) and 3327 Pa (pH 3.0, 37 °C) were measured for the absolute value of the complex shear modulus. Hydrogel swelling and degradation were influenced by the same parameters. With increasing pH and temperature the degradation time was reduced from 98 days (pH 7.4, 20 °C) to 2 days (pH 7.4, 50 °C); no degradation was observed at pH 3.0 and 5.5. Molecular modeling studies of the DA and retro-Diels-Alder (rDA) moieties revealed that hydrogel degradation occurred by rDA reaction followed by OH--catalyzed ring-opening hydrolysis of maleimide groups to unreactive maleamic acid derivatives.

Nanofibers Resulting from Cooperative Electrostatic and Hydrophobic Interactions between Peptides and Polyelectrolytes of Opposite Charge

We investigated whether cationic peptides that contain hydrophobic side chains were able to stabilize themselves via hydrophobic interactions between neighboring peptide molecules upon electrostatic binding to oppositely charged polyelectrolytes. The interaction mechanism was examined through a model system consisting of the anionic polyelectrolyte alginate and the cationic decapeptide ozarelix. The interaction resulted in the formation of highly ordered complexes that were noticeable upon visual inspection. These complexes were then investigated by microscopic techniques and shown to exhibit a branched network structure. Cryogenic-temperature transmission electron microscopy (cryo-TEM) and negative staining TEM revealed that the molecular interactions between alginate and ozarelix led to the formation of nanofibers. The rodlike nanofibers had a diameter distribution of 4-8 nm. Isothermal titration calorimetry was used to determine the thermodynamic parameters of the alginate-ozarelix interaction. The binding constant was found to be on the order of 10(6) M(-1), indicating a high binding affinity. The interaction of the peptide with the polyelectrolyte triggered profound changes in the conformation of ozarelix, which was confirmed by UV spectroscopy and circular dichroism. On the basis of these experimental results, a theoretical modeling study of the alginate-ozarelix interaction was conducted to gain a better molecular-level understanding of the complex structure. It revealed that, upon binding of ozarelix to alginate, new intermolecular and intramolecular aromatic interactions between the ozarelix molecules occurred. These interactions changed the conformation of the peptide, a modification in which the aromatic side chains played a major role. Our results indicate that the cationic peptides interact with the polyanions via electrostatic interactions, but are additionally stabilized via hydrophobic interactions. This binding mode may serve as a powerful tool to extend the duration of drug release in hydrogel drug delivery systems.

Influence of the N-terminus and the E2-loop onto the binding kinetics of the antagonist mepyramine and the partial agonist phenoprodifen to H1R

Numerous competitive radioligand binding studies revealed significant differences between human and guinea pig histamine H(1)-receptors (hH(1)R and gpH(1)R), e.g. for the partial H(1)R agonist phenoprodifen. But until now, there are only few studies with regard to binding kinetics at H(1)R. Previous studies from our group revealed an influence of the exchange of N-terminus and E2-loop between hH(1)R and gpH(1)R onto affinity of phenoprodifen to H(1)R (Strasser A, Wittmann HJ, Seifert R, J Pharmacol Exp Ther 326:783-791, 2008). The aim of this study was, therefore, to examine the impact of the N-terminus and the E2-loop on binding kinetics of the H(1)R. The wild type hH(1)R and gpH(1)R and the chimeric h(gpE2)H(1)R (E2-loogp from guinea pig) and h(gpNgpE2)H(1)R (N-terminus and E2-loop from guinea pig) were co-expressed with regulator of G-protein signaling protein RGS4 in Sf9 insect cells and kinetic binding studies were performed using the antagonist [(3)H]mepyramine as radioligand. The rate constants for association and dissociation were, in dependence of the ligand, different between hH(1)R and gpH(1)R. Furthermore, the rate constants for association at h(gpNgpE2)H(1)R were significantly different compared to hH(1)R and gpH(1)R. Molecular dynamic simulation studies detected different interactions of amino acid side chains on the extracellular surface of the receptor. Based on these findings, the influence of extracellular surface onto binding kinetics and binding affinity can be explained. Thus, the extracellular surface of G protein-coupled receptors for biogenic amines, exhibits influence onto kinetics of ligand binding, onto ligand recognition and ligand guiding into the binding pocket.

Modulation of GPCRs by monovalent cations and anions

The recent resolution of G-protein-coupled receptor (GPCR) structures in complex with Na+ bound to an allosteric modulatory site has renewed interest of the regulation of GPCRs by ions. Here, we summarise key data on ion modulation of GPCRs, obtained in pharmacological, crystallographic, mutagenesis and molecular modelling studies. We show that ion modulation is a highly complex process, involving not only cations but also, rather neglected until now, anions. Pharmacotherapeutic and toxicological aspects are discussed. We provide a mathematical framework for the analysis of ion effects. Finally, we discuss open questions in the field and future research directions. Most importantly, the in vivo relevance of the modulation of GPCR function by monovalent ions must be clarified.