Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H4 and the ion channel serotonin 5-HT3A.

A fragment library was screened against the G protein-coupled histamine H(4) receptor (H(4)R) and the ligand-gated ion channel serotonin 5-HT(3A) (5-HT(3A)R). Interestingly, significant overlap was found between H(4)R and 5-HT(3A)R hit sets. The data indicates that dual active H(4)R and 5 HT(3A)R fragments have a higher complexity than the selective compounds which has important implications for chemical genomics approaches. The results of our fragment-based library screening study illustrate similarities in ligand recognition between H(4)R and 5-HT(3A)R and have important consequences for selectivity profiling in ongoing drug discovery efforts on H(4)R and 5-HT(3A)R. The affinity profiles of our fragment screening studies furthermore match the chemical properties of the H(4)R and 5-HT(3A)R binding sites and can be used to define molecular interaction fingerprints to guide the in silico prediction of protein-ligand interactions and structure.

Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28.

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.

4-benzyl-1H-imidazoles with oxazoline termini as histamine H3 receptor agonists.

Research on the therapeutic applications of the histamine H3 receptor (H3R) has traditionally focused on antagonists/inverse agonists. In contrast, H3R agonists have received less attention despite their potential use in several disease areas. The lower availability of H3R agonists not only hampers their full therapeutic exploration, it also prevents an unequivocal understanding of the structural requirements for H3R activation. In the light of these important issues, we present our findings on 4-benzyl-1H-imidazole-based H3R agonists. Starting from two high throughput screen hits (10 and 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 H3R research.

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production.

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.

Identification of 4-(1H-imidazol-4(5)-ylmethyl)pyridine (immethridine) as a novel, potent, and highly selective histamine H(3) receptor agonist.

In this study, the piperidine ring of immepip and its analogues was replaced by a rigid heterocyclic pyridine ring. Many compounds in the series exhibit high affinity and agonist activity at the human histamine H(3) receptor. Particularly, the 4-pyridinyl analogue of immepip (1c, immethridine) is identified as a novel potent and highly selective histamine H(3) receptor agonist (pK(i) = 9.07, pEC(50) = 9.74) with a 300-fold selectivity over the closely related H(4) receptor.

Evidence for a role of p38 kinase in hypoxia-inducible factor 1-independent induction of vascular endothelial growth factor expression by sodium arsenite.

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha(-/-) mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.