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The recent crystal structure determinations of druggableclassA G protein-coupled receptors (GPCRs) have opened up excellent opportunitiesin structure-based ligand discovery for this pharmaceutically importantprotein family. We have developed and validated a customized structure-basedvirtual fragment screening protocol against the recently determinedhuman histamine H1receptor (H1R) crystal structure.The method combines molecular docking simulations with a protein–ligandinteraction fingerprint (IFP) scoring method. The optimized in silicoscreening approach was successfully applied to identify a chemicallydiverse set of novel fragment-like (≤22 heavy atoms) H1R ligands with an exceptionally high hit rate of 73%. Of the26 tested fragments, 19 compounds had affinities ranging from 10 μMto 6 nM. The current study shows the potential of in silico screeningagainst GPCR crystal structures to explore novel, fragment-like GPCRligand space. [ABSTRACT FROM AUTHOR]

The histamine H4receptor (H4R)is a G protein-coupledreceptor (GPCR) that plays an important role in inflammation. Similarto the homologous histamine H3receptor (H3R),two acidic residues in the H4R binding pocket, D3.32and E5.46, act as essential hydrogen bond acceptors ofpositively ionizable hydrogen bond donors in H4R ligands.Given the symmetric distribution of these complementary pharmacophorefeatures in H4R and its ligands, different alternativeligand binding mode hypotheses have been proposed. The current studyfocuses on the elucidation of the molecular determinants of H4R–ligand binding modes by combining (3D) quantitativestructure–activity relationship (QSAR), protein homology modeling,molecular dynamics simulations, and site-directed mutagenesis studies.We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea)derivatives to investigate H4R–ligand interactionsand ligand binding orientations. Interestingly, our studies indicatethat clobenpropit (2) itself can bind to H4R in two distinct binding modes, while the addition of a cyclohexylgroup to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H4R binding pocket. Our ligand-steered, experimentally supported proteinmodeling method gives new insights into ligand recognition by H4R and can be used as a general approach to elucidate the structureof protein–ligand complexes. [ABSTRACT FROM AUTHOR]

The histamine H3(H3R) and H4(H4R) receptors attract considerable interest from the medicinal chemistry community. Given their relatively high homology yet widely differing therapeutic promises, ligand selectivity for the two receptors is crucial. We interrogated H4R/H3R selectivities using ligands with a [1,2,3]triazole core. Cu(I)-assisted “click chemistry” was used to assemble diverse [1,2,3]triazole compounds (6a−wand 7a−f), many containing a peripheral imidazole group. The imidazole ring posed some problems in the click chemistry putatively due to Cu(II) coordination, but Boc protection of the imidazole and removal of oxygen from the reaction mixture provided effective strategies. Pharmacological studies revealed two monosubstituted imidazoles (6h,p) with 10-fold H4R/H3R selectivity. Both compounds possess a cycloalkylmethyl group and appear to target a lipophilic pocket in H4R with high steric precision. The use of the [1,2,3]triazole scaffold is further demonstrated by the notion that simple changes in spacer length or peripheral groups can reverse the selectivity toward H3R. Computational evidence is provided to account for two key selectivity switches and to pinpoint a lipophilic pocket as an important handle for H4R over H3R selectivity. [ABSTRACT FROM AUTHOR]

From a series of small fragments that was designed to probe the histamine H4receptor (H4R), we previously described quinoxaline-containing fragments that were grown into high affinity H4R ligands in a process that was guided by pharmacophore modeling. With a scaffold hopping exercise and using the same in silico models, we now report the identification and optimization of a series of quinazoline-containing H4R compounds. This approach led to the discovery of 6-chloro-N-(furan-3-ylmethyl)2-(4-methylpiperazin-1-yl)quinazolin-4-amine (VUF10499, 54) and 6-chloro-2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine (VUF10497, 55) as potent human H4R inverse agonists (pKi= 8.12 and 7.57, respectively). Interestingly, both compounds also possess considerable affinity for the human histamine H1receptor (H1R) and therefore represent a novel class of dual action H1R/H4R ligands, a profile that potentially leads to added therapeutic benefit. Compounds from this novel series of quinazolines are antagonists at the rat H4R and were found to possess anti-inflammatory properties in vivo in the rat. [ABSTRACT FROM AUTHOR]

The H4R (histamine H4 receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H4R is primarily expressed in eosinophils and mast cells and has the highest homology with the H3R. The occurrence of at least twenty different hH3R (human H3R) isoforms led us to investigate the possible existence of H4R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H4R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H4R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H4R–ligand induced signalling or constitutive activity for these H4R splice variants. However, when co-expressed with full-length H4R [H4R(390) (H4R isoform of 390 amino acids)], the H4R splice variants have a dominant negative effect on the surface expression of H4R(390). We detected H4R(390)–H4R splice varianthetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H4R splice variants were detected in various cell types and expressed at similar levels to the full-length H4R(390) mRNA in, for example, pre-monocytes. We conclude that the H4R splice variants described here have a dominant negative effect on H4R(390) functionality, as they are able to retain H4R(390) intracellularly and inactivate a population of H4R(390), presumably via hetero-oligomerization. [ABSTRACT FROM AUTHOR]

Research on the therapeutic applications of the histamine H 3receptor (H 3R) has traditionally focused on antagonists/inverse agonists. In contrast, H 3R agonists have received less attention despite their potential use in several disease areas. The lower availability of H 3R agonists not only hampers their full therapeutic exploration, it also prevents an unequivocal understanding of the structural requirements for H 3R activation. In the light of these important issues, we present our findings on 4-benzyl-1 H-imidazole-based H 3R agonists. Starting from two high throughput screen hits ( 10and 11), the benzyl side chain was altered with lipophilic groups using combinatorial and classical chemical approaches (compounds 12− 31). Alkyne- or oxazolino-substituents gave excellent affinities and agonist activities up to the single digit nM range. Our findings further substantiate the growing notion that basic ligand sidechains are not necessary for H 3R activation and reveal the oxazolino group as a hitherto unexplored functional group in H 3R research. [ABSTRACT FROM AUTHOR]

Using a previously reported flexible alignment model we have designed, synthesized, and evaluated a series of compounds at the human histamine H 4receptor (H 4R) from which 2-(4-methyl-piperazin-1-yl)-quinoxaline ( 3) was identified as a new lead structure for H 4R ligands. Exploration of the structure−activity relationship (SAR) of this scaffold led to the identification of 6,7-dichloro 3-(4-methylpiperazin-1-yl)quinoxalin-2(1 H)-one (VUF 10214, 57) and 2-benzyl-3-(4-methyl-piperazin-1-yl)quinoxaline (VUF 10148, 20) as potent H 4R ligands with nanomolar affinities. In vivo studies in the rat reveal that compound 57has significant anti-inflammatory properties in the carrageenan-induced paw-edema model. [ABSTRACT FROM AUTHOR]

Hybrid drug 1(NO-ASA) continues to attract intense research from chemists and biologists alike. It consists of ASA and a −ONO2group connected through a spacer and is in preclinical development as an antitumor drug. We report that, contrary to current beliefs, neither ASA nor NO contributes to this antitumor effect. Rather, an unsubstituted QM was identified as the sole cytotoxic agent. QM forms from 1after carboxylic ester hydrolysis and, in accordance with the HSAB theory, selectively reacts with cellular GSH, which in turn triggers cell death. Remarkably, a derivative lacking ASA and the −ONO2group is 10 times more effective than 1. Thus, our data provide a conclusive molecular mechanism for the antitumor activity of 1. Equally importantly, we show for the first time that a “presumed invisible” linker in a hybrid drug is not so invisible after all and is in fact solely responsible for the biological effect. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, the effects of endogenous histamine in the ventral hippocampus on fear memory deficits induced by scopolamine were investigated as evaluated by step-through avoidance response in adult male rats. Bilateral ventral hippocampal injection of scopolamine (i.h., 2, 5 μg/site) significantly produced depressant effects on the active avoidance response in a dose-dependent manner. Histamine H3-antagonist clobenpropit (5, 10 μg/site) significantly ameliorated the fear memory deficits induced by scopolamine in a dose-dependent manner. Its procognitive effect was completely antagonized by immepip (10 μg/site), a selective histamine H3-agonist. Both histamine H1-antagonist pyrilamine and H2-antagonist cimetidine, also inhibited the procognitive effects of clobenpropit. Additionally, the procognitive effects of clobenpropit on the fear memory deficits induced by scopolamine were significantly potentiated by intraperitoneal (i.p.) injection of histidine, a precursor of histamine, but markedly reversed by i.h. injection of α-fluoromethylhistidine (FMH, 10 μg/site), a selective and potent histidine decarboxylase inhibitor. It is concluded that the increased endogenous histamine release in the ventral hippocampus ameliorates the scopolamine-induced fear memory deficits, and its action is mainly mediated by histamine presynaptic H3-receptors and postsynaptic H1- and H2-receptors. [Copyright &y& Elsevier]