Abolishing Dopamine D2long/D3 Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H2 Receptor Agonists.

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H2 receptor (H2R) agonists, but their applicability as pharmacological tools to elucidate the largely unknown H2R functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D2-like receptors (especially to the D3R). To improve the selectivity, a series of novel carbamoylguanidine-type ligands containing various heterocycles, spacers, and side residues were rationally designed, synthesized, and tested in binding and/or functional assays at H1-4 and D2long/3 receptors. This study revealed a couple of selective candidates (among others 31 and 47), and the most promising ones were screened at several off-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42, and 7.35) are responsible for the different affinities at the H2- and D2long/3-receptors. These results provide a solid base for the exploration of the H2R functions in the brain in further studies.

Structure-Activity Relationships and Computational Investigations into the Development of Potent and Balanced Dual-Acting Butyrylcholinesterase Inhibitors and Human Cannabinoid Receptor 2 Ligands with Pro-Cognitive in Vivo Profiles.

The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCB2R) represent promising targets for pharmacotherapy in the later stages of Alzheimer's disease. We merged pharmacophores for both targets into small benzimidazole-based molecules, investigated SARs, and identified several dual-acting ligands with a balanced affinity/inhibitory activity and an excellent selectivity over both hCB1R and hAChE. A homology model for the hCB2R was developed based on the hCB1R crystal structure and used for molecular dynamics studies to investigate binding modes. In vitro studies proved hCB2R agonism. Unwanted µ-opioid receptor affinity could be designed out. One well-balanced dual-acting and selective hBChE inhibitor/hCB2R agonist showed superior in vivo activity over the lead CB2 agonist with regards to cognition improvement. The data shows the possibility to combine a small molecule with selective and balanced GPCR-activity/enzyme inhibition and in vivo activity for the therapy of AD and may help to rationalize the development of other dual-acting ligands.

Binding Kinetics and Pathways of Ligands to GPCRs.

Previously, drugs were developed focusing on target affinity and selectivity. However, it is becoming evident that the drug-target residence time, related to the off-rate, is an important parameter for successful drug development. The residence time influences both the on-rate and overall effectiveness of drugs. Furthermore, ligand binding is now appreciated to be a multistep process because metastable and/or intermediate binding sites in the extracellular region have been identified. In this review, we summarize experimental ligand-binding data for G-protein-coupled receptors (GPCRs), and their binding pathways, analyzed by molecular dynamics (MD). The kinetics of drug binding to GPCRs are complex and depend on several factors, including charge distribution on the receptor surface, ligand-receptor interactions in the binding channel and the binding site, or solvation.

Competitive association binding kinetic assays: a new tool to detect two different binding orientations of a ligand to its target protein under distinct conditions?

Within the last years, for several ligands, binding to G protein-coupled receptors or other target proteins, a binding of the ligand in two different orientations is described. One appropriate experimental technique to detect two different binding orientations is the crystallization of the ligand-protein-complex, but crystallization and subsequent X-ray analysis do not belong to the routine methods. By traditional competitive radioligand equilibrium binding assays, it is not possible to detect or to distinguish between two different binding orientations, but there is a possibility to identify two different binding orientations by performing kinetic competitive radioligand-binding assays. To study the limitations of this new technique, the related differential equations were defined and solved numerically for 8 different sets of rate constants, also considering an experimental error up to ~10%. In principal, the kinetic competitive radioligand binding assay is a suitable technique to detect two different ligand binding orientations. However, the present study shows that this is only possible under distinct conditions: (1) the rate constants of dissociation for both binding orientations of the cold ligand should at least be >> 10-fold different to each other and (2) the experimental error should be as small as possible. Although there are some limitations for the experimental usability of this method, it is worthwhile to perform kinetic competitive binding assays, especially if there are hints for two binding orientations of a ligand, e.g. based on molecular modelling studies.

Molecular Modelling Approaches for the Analysis of Histamine Receptors and Their Interaction with Ligands.

Several experimental techniques to analyse histamine receptors are available, e.g. pharmacological characterisation of known or new compounds by different types of assays or mutagenesis studies. To obtain insights into the histamine receptors on a molecular and structural level, crystal structures have to be determined and molecular modelling studies have to be performed. It is widely accepted to generate homology models of the receptor of interest based on an appropriate crystal structure as a template and to refine the resulting models by molecular dynamic simulations. A lot of modelling techniques, e.g. docking, QSAR or interaction fingerprint methods, are used to predict binding modes of ligands and pharmacological data, e.g. affinity or even efficacy. However, within the last years, molecular dynamic simulations got more and more important: First of all, molecular dynamic simulations are very helpful to refine the binding mode of a ligand to a histamine receptor, obtained by docking studies. Furthermore, with increasing computational performance it got possible to simulate complete binding pathways of ions or ligands from the aqueous extracellular phase into the allosteric or orthosteric binding pocket of histamine receptors.

Dibenzo[b,f][1,4]oxazepines and dibenzo[b,e]oxepines: Influence of the chlorine substitution pattern on the pharmacology at the H1R, H4R, 5-HT2AR and other selected GPCRs.

Inspired by VUF6884 (7-Chloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine), reported as a dual H1/H4 receptor ligand (pKi: 8.11 (human H1R (hH1R)), 7.55 (human H4R (hH4R))), four known and 28 new oxazepine and related oxepine derivatives were synthesised and pharmacologically characterized at histamine receptors and selected aminergic GPCRs. In contrast to the oxazepine series, within the oxepine series, the new compounds showed high affinity to the hH1R (pKi: 6.8-8.7), but no or moderate affinity to the hH4R (pKi:≤5.3). For one oxepine derivative (1-(2-Chloro-6,11-dihydrodibenzo[b,e]oxepin-11-yl)-4-methylpiperazine), the enantiomers were separated and the R-enantiomer was identified as the eutomer at the hH1R (pKi: 8.83 (R), 7.63 (S)) and the guinea-pig H1R (gpH1R) (pKi: 8.82 (R), 7.41 (S)). Molecular dynamic studies suggest that the tricyclic core of the compounds is bound in a similar mode into the binding pocket, as described for doxepine in the hH1R crystal structure. Moreover, docking studies of all oxepine derivatives at the hH1R indicate that the oxygen and the position of the chlorine in the tricyclic core determines, if the R- or the S-enantiomer is the eutomer. For some of the oxazepines and oxepines the affinity to other aminergic GPCRs is in the same range as to hH1R or hH4R, thus, those compounds have to be classified as dirty drugs. However, one oxazepine derivative (3,7-Dichloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine was identified as dual hH1/h5-HT2A receptor ligand (pKi: 9.23 (hH1R), 8.74 (h5-HT2AR), ≤7 at other analysed GPCRs), whereas one oxepine derivative (1-(3,8-Dichloro-6,11-dihydrodibenzo[b,e]oxepin-11-yl)-4-methylpiperazine) was identified as selective hH1R antagonist (pKi: 8.44 (hH1R), ≤6.7 at other analyzed GPCRs). Thus, the pharmacological results suggest that the oxazepine/oxepine moiety and additionally the chlorine substitution pattern toggles receptor selectivity and specificity.

Conformational Restriction and Enantioseparation Increase Potency and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor Agonists.

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H4 receptor (hH4R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hHxR (x: 1-4) subtypes on Sf9 cell membranes (radioligand binding, [(35)S]GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H4R (HEK-293 cells). The most potent H4R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R, >100-fold selectivity; H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([(35)S]GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.

2,4-Diaminopyrimidines as dual ligands at the histamine H1 and H4 receptor-H1/H4-receptor selectivity.

Distinct diaminopyrimidines, for example, 4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine are histamine H4 receptor (H4R) antagonists and show high affinity to the H4R, but only a moderate affinity to the histamine H1 receptor (H1R). Within previous studies it was shown that an aromatic side chain with a distinct distance to the basic amine and aromatic core is necessary for affinity to the human H1R (hH1R). Thus, a rigid aminopyrimidine with a tricyclic core was used as a lead structure. There, (1) the flexible aromatic side chain was introduced, (2) the substitution pattern of the pyrimidine core was exchanged and (3) rigidity was decreased by opening the tricyclic core. Within the present study, two compounds with similar affinity in the one digit µM range to the human H1R and H4R were identified. While the affinity at the hH1R increased about 4- to 8-fold compared to the parent diaminopyrimidine, the affinity to the hH4R decreased about 5- to 8-fold. In addition to the parent diaminopyrimidine, two selected compounds were docked into the H1R and H4R and molecular dynamic studies were performed to predict the binding mode and explain the experimental results on a molecular level. The two new compounds may be good lead structures for the development of dual H1/H4 receptor ligands with affinities in the same range.

Aminobenzimidazoles and Structural Isomers as Templates for Dual-Acting Butyrylcholinesterase Inhibitors and hCB2 R Ligands To Combat Neurodegenerative Disorders.

A pharmacophore model for butyrylcholinesterase (BChE) inhibitors was applied to a human cannabinoid subtype 2 receptor (hCB2 R) agonist and verified it as a first-generation lead for respective dual-acting compounds. The design, synthesis, and pharmacological evaluation of various derivatives led to the identification of aminobenzimidazoles as second-generation leads with micro- or sub-micromolar activities at both targets and excellent selectivity over hCB1 and AChE, respectively. Computational studies of the first- and second-generation lead structures by applying molecular dynamics (MD) on the active hCB2 R model, along with docking and MD on hBChE, has enabled an explanation of their binding profiles at the protein levels and opened the way for further optimization. Dual-acting compounds with "balanced" affinities and excellent selectivities could be obtained that represent leads for treatment of both cognitive and pathophysiological impairment occurring in neurodegenerative disorders.

Structure-Dependent Deconjugation of Flavonoid Glucuronides by Human ß-Glucuronidase - In Vitro and In Silico Analyses.

Flavonoid glycosides are extensively metabolized to glucuronidated compounds after oral intake. Recently, a cleavage of quercetin glucuronides by ß-glucuronidase has been found. To characterize the deglucuronidation reaction and its structural prerequisites among the flavonoid subtypes more precisely, four flavonol glucuronides with varying glucuronidation positions, five flavone 7-O-glucuronides with varying A- and B-ring substitution as well as one flavanone- and one isoflavone-7-O-glucuronide were analyzed in a human monocytic cell line. Investigation of the deglucuronidation rates by HPLC revealed a significant influence of the glucuronidation position on enzyme activity for flavonols. Across the flavonoid subtypes, the C-ring saturation also showed a significant influence on deglucuronidation, whereas A- and B-ring variations within the flavone-7-O-glucuronides did not affect the enzymes' activity. Results were compared to computational binding studies on human ß-glucuronidase. Additionally, molecular modeling and dynamic studies were performed to obtain detailed insight into the binding and cleavage mode of the substrate at the active site of the human ß-glucuronidase.

Binding pathway of histamine to the hH4R, observed by unconstrained molecular dynamics.

Histamine binds with high affinity to the human histamine H4 receptor (hH4R). We are the first to examine the complete binding pathway of histamine from the extracellular side to the orthosteric binding site of the hH4R by means of unconstrained molecular dynamic simulation. Furthermore, the simulations show that the positively charged amine moiety of the histamine interacts electrostatically with the highly conserved Asp(3.32), while the imidazole moiety forms a hydrogen bond interaction with Glu(5.46) and Gln(7.42).

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.

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.

Sodium binding to hH3R and hH 4R--a molecular modeling study.

Several aminergic GPCRs, e.g., the human histamine H3-receptor (hH3R) are sensitive to sodium ions. Based on these experimental results, including site directed mutagenesis studies, a sodium binding pocket near to the highly conserved Asp2.50 was suggested. Recently, in the crystallized adenosine A2A receptor (4EIY), a sodium ion was found in a pocket, coordinated by Asp52, Ser91, and three water molecules. Despite high homology in amino acid sequence between hH3R and hH4R, pharmacological studies revealed that the hH4R is--in contrast to hH3R--not sensitive to sodium ions. In order to obtain a deeper insight onto the differences in sodium sensitivity between hH3R and hH4R, we performed molecular modelling studies, including molecular dynamic simulations and calculation of Gibbs energy of solvation. The results of the modeling studies suggested that the amino acid at position 7.42 influences sodium binding to aminergic GPCRs in different ways. A comparison of the amino acids forming the sodium binding channel between the ligand binding pocket and the sodium binding pocket of all human aminergic GPCRs showed an 80% occurrence of glycine--in contrast to hH3R and hH4R. The Gln7.42 at hH4R disrupts a water chain, connecting the Asp3.32 of the orthosteric binding site and the Asp2.50 of the allosteric binding site. Besides, the oxygen of the glutamine side chain stabilizes the interaction of the sodium ion with the Asp3.32. Thus, the binding of the sodium into the allosteric binding site might be hindered kinetically.

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 Alzheimer's 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.

Pharmacological profile of astemizole-derived compounds at the histamine H1 and H4 receptor--H1/H4 receptor selectivity.

Astemizole, a H1R antagonist shows high affinity to the histamine H1 receptor but only a moderate affinity to the histamine H4 receptor. This study aims to modify the astemizole to keep high affinity to the histamine H1 receptor and to increase affinity to the histamine H4 receptor. Therefore, 13 astemizole-derived compounds and astemizole-JNJ7777120-derived hybrid compounds were synthesized and pharmacologically characterized at the histamine H1 and H4 receptors. The new compounds show affinity to the histamine H1 receptor in the pK i range from 5.3 to 8.8, whereas the affinity of these compounds to the histamine H4 receptor was surprisingly rather low (pK i from 4.4 to 5.6). Three representative compounds were docked into the histamine H1 receptor and molecular dynamic studies were performed to explain the binding mode and the experimental results on a molecular level. Furthermore, taking into account the binding mode of compounds with high affinity to the histamine H4 receptor, a H1/H4-pharmacophore hypothesis was developed.

Mathematical analysis of the sodium sensitivity of the human histamine H3 receptor.

PURPOSE: It was shown by several experimental studies that some G protein coupled receptors (GPCR) are sensitive to sodium ions. Furthermore, mutagenesis studies or the determination of crystal structures of the adenosine A2A or δ-opioid receptor revealed an allosteric Na(+) binding pocket near to the highly conserved Asp(2.50). Within a previous study, the influence of NaCl concentration onto the steady-state GTPase activity at the human histamine H3 receptor (hH3R) in presence of the endogenous histamine or the inverse agonist thioperamide was analyzed. The purpose of the present study was to examine and quantify the Na(+)-sensitivity of hH3R on a molecular level. METHODS: To achieve this, we developed a set of equations, describing constitutive activity and the different ligand-receptor equilibria in absence or presence of sodium ions. Furthermore, in order to gain a better understanding of the ligand- and Na(+)-binding to hH3R on molecular level, we performed molecular dynamic (MD) simulations. RESULTS: The analysis of the previously determined experimental steady-state GTPase data with the set of equations presented within this study, reveals that thioperamide binds into the orthosteric binding pocket of the hH3R in absence or presence of a Na(+) in its allosteric binding site. However, the data suggest that thioperamide binds preferentially into the hH3R in absence of a sodium ion in its allosteric site. These experimental results were supported by MD simulations of thioperamide in the binding pocket of the inactive hH3R. Furthermore, the MD simulations revealed two different binding modes for thioperamide in presence or absence of a Na(+) in its allosteric site. CONCLUSION: The mathematical model presented within this study describes the experimental data regarding the Na(+)-sensitivity of hH3R in an excellent manner. Although the present study is focused onto the Na(+)-sensitivity of the hH3R, the resulting equations, describing Na(+)- and ligand-binding to a GPCR, can be used for all other ion-sensitive GPCRs.

Molecular modeling studies give hint for the existence of a symmetric hß2R-Gαßγ-homodimer.

Several experimental studies suggest that GPCR dimers or oligomers may play an important role in signal transduction. In 2011 the crystal structure of a hß2R-Gαßγ-complex was published and crystal structures of GPCR dimers are known. But until now, no crystal structure of a GPCR dimer including the Gαßγ-complex is available. In order to obtain detailed insights into interactions within hß2R dimers including the Gαßγ-complex we performed a potential-energy-surface scan in order to identify favored asymmetric and symmetric hß2R-Gαßγ-homodimers. This potential energy surface scan suggests, besides the existence of asymmetric dimers, the existence of a symmetric hß2R-Gαßγ-homodimer with a TM I/VII-contact. A subsequent 20 ns MD simulation of the symmetric homodimer revealed large asymmetric conformational changes of both hß2Rs, especially regarding TM VII and the interaction network between Asp(2.50), Val(7.44), Ser(7.46) and Tyr(7.43). Since similar conformational changes were not observed during the molecular dynamic simulation of the monomeric hß2R-Gαßγ-complex, it may be suggested that the conformational changes in the symmetric homodimer are related to the presence of the second hß2R-Gαßγ-complex. Due to the limitations of simulation time, conformational changes within a time scale of µs or ms may of course not be observed. However, the detected conformational changes, especially in TM VII, correspond to minima on the potential energy surface and thus, this study gives new insights into GPCR dimers on molecular level and furthermore, gives suggestions for site-directed mutagenesis studies.

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.

hß2R-Gαs complex: prediction versus crystal structure--how valuable are predictions based on molecular modeling studies?

In 2010, we predicted two models for the hß(2)R-Gα(s) complex by combining the technique of homology modeling with a potential energy surface scan, since a complete crystal structure of the hß(2)R-Gα(s) complex was not available. The crystal structure of opsin co-crystallized with part of the C-terminus of Gα (3DQB) was used as a template to model the hß(2)R, whereas the crystal structure of Gα (1AZT) was used as a template to model Gα(s). Utilizing a potential energy surface scan between hß(2)R and Gα(s), a six-dimensional potential energy surface was obtained. Two significant minimum regions were located on this surface, and each was associated with a distinct hß(2)R-Gα(s) complex, namely model I and model II [Straßer A, Wittmann H-J (2010) J Mol Model 16:1307-1318]. The crystal structure of the hß(2)R-Gα(s)ßγ complex has recently been published. Thus, the aim of the current study was, on the one hand, to compare our predicted structures with the true crystal structure, and on the other to discuss the question: how valuable are predictions based on molecular modeling studies?