In this study, we replaced the basic amine function of the known histamine H(3) receptor agonists imbutamine or immepip with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human H(3) receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed an increased selectivity for the human H(3) receptor over the human H(4) receptor, but none of the compounds in this series possess increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-imidazol-4-yl)-pentan-1-ol, is the most potent histamine H(3) receptor agonist (pK(i) = 8.0 and pEC(50) = 8.1) with a 320-fold selectivity at the human H(3) receptor over the human H(4) receptor.

The histamine H2 receptor belongs to the large family of G protein-coupled receptors (GPCRs) and is positively coupled to adenylate cyclase. This chapter discusses a variety of experimental approaches that can be used to determine the constitutive activity of the H2 receptor or other Gs-coupled receptors. With the aid of different heterologous expression techniques, constitutive activity of the H2 receptor is detected easily in a wide variety of cell lines. Especially at high levels of receptor expression, the increase in basal CAMP production can be observed easily (either direct or via a reporter gene assay). Although modulation of GPCR expression is an important factor in the detection of constitutive signaling, the level of GPCR expression is also an important determinant for the extent of the constitutive responsiveness. The expression levels of the various partners in the GPCR signaling pathway also determine the final agonist-independent output. For the Gq-coupled muscarinic m3 receptor, overexpression of Gαq increases the basal activation of signal transduction pathways.

New molecular modeling tools were developed to construct a qualitative pharmacophore model for histamine H3 receptor antagonists. The program SLATE superposes ligands assuming optimum hydrogen bond geometry. One or two ligands are allowed to flex in the procedure, thereby enabling the determination of the bioactive conformation of flexible H3 antagonists. In the derived model, four hydrogen-bonding site points and two hydrophobic pockets available for binding antagonists are revealed. The model results in a better understanding of the structure-activity relationships of H3 antagonists. To validate the model, a series of new antagonists was synthesized. The compounds were designed to interact with all four hydrogen-bonding site points and the two hydrophobic pockets simultaneously. These ligands have high H3 receptor affinity, thereby illustrating how the model can be used in the design of new classes of H3 antagonists.

We describe the use of recombinant Semliki Forest virus (SFV) vectors for efficient expression of the rat histamine H(2) (rH(2)) receptor in COS-7 (African green monkey kidney cells) cells. Recombinant SFV-infected COS-7 cells express the histamine rH(2) receptor in a time-dependent fashion with a maximum expression level of 50 pmol mg(-1) after 40 h. SFV-mediated histamine rH(2) receptor expression shows similar pharmacological properties as the receptor expressed transiently or stably in mammalian cells. In addition, we demonstrate the pharmacological and functional characterisation of the D(115)N mutated histamine rH(2) receptor. It has been shown that the D(115)N mutation renders the receptor constitutively active and structurally unstable. The rapid onset of and high maximal expression levels obtained from SFV-infected COS-7 cells enabled us to characterise this mutant receptor. We prove that recombinant SFV vectors are powerful tools for heterologous expression of G-protein-coupled receptors and that one can achieve both the high-level gene expression described for baculovirus-infected insect cells and the use of mammalian cells as hosts.

Following the cloning of the genes encoding histamine H1 and H2 receptors in 1991 ( 1 Gantz I. et al. Molecular cloning of a gene encoding the histamine H2 receptor. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 429-433 Crossref PubMed Scopus (238) Google Scholar , 2 Yamashita M. et al. Expression cloning of a cDNA encoding the bovine histamine H1 receptor. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11515-11519 Crossref PubMed Scopus (230) Google Scholar ), the discovery of additional histamine receptor subtypes was expected. However, in contrast to the receptor families for dopamine or 5-HT, the introduction of molecular biology has thus far not led to the identification of new histamine receptor subtypes. Moreover, despite considerable efforts to clone the H3 receptor, details of the molecular structure of this therapeutically interesting receptor subtype were lacking until June of last year 3 Lovenberg T.W. et al. Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 1999; 55: 1101-1107 Crossref PubMed Scopus (712) Google Scholar . Because homology-based approaches and expression-cloning strategies carried out by various laboratories were not successful, some speculated that, like the 5-HT3 receptor, the H3 receptor might not be a G-protein-coupled receptor (GPCR). Finally, information from a private expressed sequence tag (EST) database led to the identification of the H3 receptor as a GPCR ( 3 Lovenberg T.W. et al. Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 1999; 55: 1101-1107 Crossref PubMed Scopus (712) Google Scholar ). Lovenberg and colleagues identified an EST (GPCR97) that possessed 35% amino acid homology with the seventh transmembrane domain (TM7) of α2-adrenoceptors. Predominant expression of the partial GPCR97 sequence in the CNS prompted Lovenberg et al. to clone the full-length GPCR97 sequence (GenBank AF140538) from a size-selected human thalamic cDNA library 3 Lovenberg T.W. et al. Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 1999; 55: 1101-1107 Crossref PubMed Scopus (712) Google Scholar . The presence of a conserved aspartate residue in TM3 (Asp114) identified GPCR97 as a member of the GPCR family of amine transmitter systems. Signal transduction assays and radioligand binding studies revealed that GPCR97 was, in fact, the Gi-coupled histamine H3 receptor 3 Lovenberg T.W. et al. Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 1999; 55: 1101-1107 Crossref PubMed Scopus (712) Google Scholar .

Various approaches to the synthesis of all four stereoisomers of 2-(1-H-imidazol-4-yl) cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct transcyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H-3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD(2) = 7.1; alpha = 0.75) and on guinea pig jejunum (pD(2) = 6.6; alpha = 0.75). Its enantiomer, (1R,2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show we ak activity on H-1 and H-2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H-3 receptor ligands.

Various approaches to the synthesis of all four stereoisomers of 2-(1-H-imidazol-4-yl) cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct transcyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H-3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD(2) = 7.1; alpha = 0.75) and on guinea pig jejunum (pD(2) = 6.6; alpha = 0.75). Its enantiomer, (1R,2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show we ak activity on H-1 and H-2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H-3 receptor ligands.

Abstract: Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine‐induced increase in cAMP and the cimetidine‐induced reduction of the basal cAMP level with apparent KB values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long‐term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an upregulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.

About 3 kb of the promoter region of the gene encoding cytochrome P-450 2B2 (CYP2B2) in the rat were sequenced and searched for potential cis-acting elements. Apart from putative binding sites for (liver-specific) protein factors, a region showing homology with the LINE 1 retrotransposon element was also found. Three proximal promoter fragments, encompassing nucleotides -579 to -372, -372 to -211, and -211 to +1, respectively, were shown to contain binding sites for multiple protein factors by bandshift analyses. The strongest protein-binding element, designated BRE (basic regulatory element), occurs between -103 to -66. Its structure is very similar to a negative control element in the murine cmyc promoter and displays a composite feature having a tandemly repeated sequence homology with the BTE (basic transcription element; Yanagida et al., 1990) separated by a CCAAA-box. The use of a deletion series of this template in in vitro transcription assays, provided evidence that the BRE serves as a major cis-acting element in the (regulated) transcription activation of the CYP2B2 gene.