Shining light on the histamine H receptor: Synthesis of carbamoylguanidine-type agonists as a pharmacological tool to study internalization.

So far, only little is known about the internalization process of the histamine H receptor (HR). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type HR agonists containing various fluorophores, heterocycles, and linkers (28-40) was synthesized.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations.

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H and H receptors (HR and HR) are prominent members of Gs- and Gi-coupled GPCRs.

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors.

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such "invasive" techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood.

Abolishing Dopamine D/D Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H Receptor Agonists.

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H receptor (HR) agonists, but their applicability as pharmacological tools to elucidate the largely unknown HR functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D-like receptors (especially to the DR). 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 H and D receptors. This study revealed a couple of selective candidates (among others and ), and the most promising ones were screened at several off-target receptors, showing good selectivities.

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes.

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([S]GTPγS, [γ-P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands.

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 (hCBR) 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 hCBR and hAChE. A homology model for the hCBR was developed based on the hCBR crystal structure and used for molecular dynamics studies to investigate binding modes.

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.

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.

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.

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

Inspired by VUF6884 (7-Chloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine), reported as a dual H/H receptor ligand (pK: 8.11 (human HR (hHR)), 7.55 (human HR (hHR))), four known and 28 new oxazepine and related oxepine derivatives were synthesised and pharmacologically characterized at histamine receptors and selected aminergic GPCRs.

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).

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.

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.

Structure-Dependent Deconjugation of Flavonoid Glucuronides by Human β-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 β-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.

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.

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.

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).

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.

Loratadine and analogues: discovery and preliminary structure-activity relationship of inhibitors of the amino acid transporter B(0)AT2.

B(0)AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds.

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.

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.

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.

Molecular modeling studies give hint for the existence of a symmetric hβ₂R-Gαβγ-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 hβ2R-Gαβγ-complex was published and crystal structures of GPCR dimers are known. But until now, no crystal structure of a GPCR dimer including the Gαβγ-complex is available.

Lysis matters: red cell lysis with FACS Lyse affects the flow cytometric enumeration of circulating leukemic blasts.

The whole blood lysis method has become a standard procedure to remove red cells prior to immunophenotypic analysis of leukocytes. In the present study we investigated the influence of four different lysis protocols on the flow cytometric recovery of leukemic blasts. 32 blast cells containing blood samples were stained with anti-CD45 and anti-CD34 monoclonal antibody combinations.

Molecular and cellular analysis of human histamine receptor subtypes.

The human histamine receptors hH(1)R and hH(2)R constitute important drug targets, and hH(3)R and hH(4)R have substantial potential in this area. Considering the species-specificity of pharmacology of H(x)R orthologs, it is important to analyze hH(x)Rs. Here, we summarize current knowledge of hH(x)Rs endogenously expressed in human cells and hH(x)Rs recombinantly expressed in mammalian and insect cells.