Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms

The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.     

Malaria: use of restriction endonuclease digestion and mutation-specific PCR for antifolate resistance isolate detection

Antifolate resistance isolates of Plasmodium falciparum in the blood of 56 patients was investigated by using PCR technology. DNA was extracted with three different methods from parasite lysate by phenol-chloroform, or from whole blood and from blood collected onto dry filter paper, by chelex-100. The expected 727-bp PCR product was obtained in all samples extracted by chelex-100, while three samples prepared by phenol-chloroform failed to show any amplified product. The crucial point mutation within the dhfr gene leading to pyrimethamine and cycloguanil resistance is localised in an Alul recognition site. Thus, the 727-bp PCR product was submitted to endonuclease digestion. Fifty out of the 56 blood samples analysed yielded the two expected restriction fragments and an undigested 727-bp band. These 50 samples likely represent mixed infection as also confirmed the specific mutation PCR. The six undigested samples amplify a 339-bp fragment using a nested PCR-specific for pyrimethamine resistance mutation. Our results show that, the rapid DNA extraction from blood using chelex-100 and the PCR endonuclease assay can be efficiently used for accurate chemosensitivity analysis in the field.     

Fluorinated non-imidazole histamine H3 receptor antagonists

Fluorine substituents have become a widespread and important component in drug design and development. Here, the synthesis of fluorine containing compounds and some corresponding precursor molecules are presented for potential isotope labelling as well as their data obtained with in vitro and in vivo screenings. The compounds vary in the basic centres (piperidine or pyrrolidine) and are fluoro substituted in different positions of the basic alicyclic moiety. Pharmacological evaluation resulted in ligands with high affinities at hH₃ receptor in the nanomolar and subnanomolar concentration range and some with high antagonist in vivo potencies.     

Non-imidazole heterocyclic histamine H3 receptor antagonists

Continued exploration of the SAR around the lead imidazopyridine histamine H(3) antagonist 1 has led to the discovery of several related series of heterocyclic histamine H(3) antagonists. The synthesis and SAR of indolizine, indole and pyrazolopyridine based compounds are now described.     

Diagnosis of mycobacterial infections by PCR and restriction enzyme digestion

A method for the diagnosis of mycobacterial infections by PCR amplification followed by selective restriction enzyme digestion of the PCR product was developed. The amplified DNA sequence used in this study occurs within the gene encoding for the mycobacterial 65 kDa heat shock protein (Hance et al. 1989), which is found in all mycobacteria. However, there are minute differences in the amplified sequence from the Mycobacterium tuberculosis complex compared with the corresponding sequence from the Mycobacterium avium complex. These differences made it possible to rapidly identify to which mycobacterial complex a particular sample belonged by restriction enzyme digestion of the PCR product. A total of 66 samples were tested and all of them were correctly identified. This and similar methods should provide a sensitive, specific and rapid (within 12 h) way of diagnosing mycobacterial infections to the species level.     

Acidic elements in histamine H3 receptor antagonists

Antagonists of the human histamine H₃ receptor (hH₃R) often contain a second basic moiety, which is well known to boost affinity on this histamine receptor subtype. Here, we prepared compounds with acidic moieties of different pK_a values to figure out that the hH₃R tolerates these functionalities when added to a common pharmacophore blueprint. Depending on the acidic, electronic and steric features the designed ligands showed hH₃R affinities in the nanomolar concentration range. Additionally, selected ligands were tested but failed as dual acting hH₃R/hPPAR (human peroxisome proliferator-activated receptor) ligands.     

组胺H3受体研究进展

组胺H3受体作为突触前自身受体和异身受体,广泛分布于中枢和外周组织,抑制组胺的释放和合成,调节多种神经递质的释放,组胺H3受体是G蛋白偶联受体家族成员,激活后由G蛋白介导,通过调控N型Ca^2 通道,产生生物学效应,组胺H3受体在中枢和外周器官有着重要的生理功能,对心功能,胃酸分泌,觉醒的睡眠,认知和记忆,惊厥抽搐等都有调节作用。     

Early detection of Schistosoma mansoni infection by touchdown PCR in a mouse model

A detection assay for Schistosoma mansoni DNA in mouse serum samples based on touchdown PCR was developed and evaluated. The serum samples could be assayed directly without the need to extract DNA. No cross reactions between S. mansoni and related species inducing human schistosomiasis were observed. After the infection, mouse sera and feces were collected for 8 weeks. Anti-worm antigen IgG and anti-soluble egg antigen IgG were detected in the sera at 6 weeks post-infection by ELISA. The parasite's eggs were detected in the feces at 8 weeks. In contrast, S. mansoni DNA was detected in the sera at 2 weeks post-infection. These data suggest that touchdown PCR is a potential tool for the early diagnosis of S. mansoni infection.     

Genetic and pharmacological aspects of histamine H3 receptor heterogeneity

Histaminergic H3 receptors modulate the release of neurotransmitters within the CNS and periphery. Ligands for these receptors have potential clinical utility in a variety of disease states. However, the pharmacological characteristics of these receptors have been enigmatic for more than a decade because of the diversity of pharmacological effects observed with the limited number of heretofore-available compounds. Recent cloning of the H3 receptor has revealed interspecies differences in the protein sequences in key regions, the existence of splice variants that differ in composition between species, and potential differences in signal transduction processes between either different tissues and/or species. This review attempts to summarize these findings within the context of the molecular biological and pharmacological data accumulated to date. Also, we suggest a nomenclature strategy to reduce potential confusion that has arisen from different naming systems used by various investigators. While some facets of this genetic and pharmacological diversity help to rationalize various aspects of H3 receptor heterogeneity, there remains an insufficient repertoire of selective ligands, assays, or other measures to completely resolve all components of this diversity. The promise of newly available tools to further explore H3 receptor function may provide the insight to bring the promised clinical potential of H3 receptor ligands to realization.     

Rasagiline derivatives combined with histamine H3 receptor properties

The irreversible monoamine oxidase B (MAO B) inhibitor rasagiline has been described with multiple disease modifying effects in vitro on models of Parkinson’s disease. The combination of this established drug to recently developed histamine H₃ receptor (H₃R) antagonist elements gives new impetus to the design of multitargeting ligands. Surprisingly, the 5-substituted 3-piperidinopropyloxy rasagiline derivative 1 was more potent on both targets than its 6-substituted isomer. It showed nanomolar affinities at the desired targets (MAO B IC₅₀ = 256 nM; hH₃R K_i = 2.6 nM) with a high preference over monoamine oxidase A (MAO A) and negligible affinity at histamine H₁, H₄, dopamine D₂, D₃ receptors or acetyl-/butyrylcholinesterases.     

The rat H3 receptor: gene organization and multiple isoforms

Genomic DNA analysis revealed that the coding region of the rat histamine H3 receptor comprises three exons interrupted by two introns of approximately 1 kb each. Several H3 receptor mRNA variants were identified by PCR and cDNA cloning and sequencing. Four variants generated by pseudo-intron retention/deletion at the level of the third intracellular loop were designated H3(445), H3(413), H3(410), and H3(397), according to the length of their deduced amino acid sequence and display differential tissue expression. When expressed in CHO-K1 or Cos-1 cells, the H3(445), H3(413), and H3(397) were found to generate specific 125I iodoproxyfan binding of similar pharmacological profile. In addition, we identified two short variants, termed H3(nf1) and H3(nf2), which correspond to frame shift and stop codon interposition, respectively, and are presumably nonfunctional, among which H3(nf2) displays brain expression similar to that of the longer isoforms.     

Fluorescent non-imidazole histamine H3 receptor ligands with nanomolar affinities

Omega-piperidinoalkanamine derivatives with fluorescent moieties (2-cyanoisoindol-1-yl, 7-nitrobenzofurazan-4-yl) have been synthesized starting from piperidine in three steps. The compounds display moderate to good histamine hH(3) receptor affinities with K(i) values ranging from 178 to 11nM. The new compounds may act as tools for identification and understanding of the binding site on the histamine H(3) receptor.     

Changes in histamine H3 receptor responsiveness in mouse brain

Changes in various histamine (HA) H3 receptor-mediated responses and H3 receptor binding in brain were investigated in mice receiving single or repeated administration of ciproxifan, a potent brain-penetrating and selective H3 receptor antagonist. Blockade of the H3 autoreceptor was nearly as effective in enhancing levels of tele-methylhistamine (t-MeHA), a major HA metabolite, in brain areas when ciproxifan was administered once either at 7 a.m. or 8 p.m., in spite of the large differences of basal levels at these two phases of the circadian cycle. Blockade after a single ciproxifan administration was, however, followed by a transient decrease in striatal t-MeHA levels, possibly reflecting rapid development of autoreceptor hypersensitivity. Following a 5-day administration of ciproxifan and a 2-day drug-free period, basal t-MeHA levels were significantly decreased (approximately -20%) in three brain areas, and the ED50 values of the drug to enhance t-MeHA levels were increased by 5-15 times without significant change in maximal response, indicating that H3 autoreceptor hypersensitivity had developed. However, in synaptosomes from the cerebral cortex of these animals, the H3 receptor-mediated inhibition of K+-induced [3H]HA release was not significantly modified. Subchronic administration of ciproxifan for 10 days also resulted in an increased binding of [125I]iodoproxyfan to the H3 receptor of striatal and hypothalamic membranes by 40-54%. Hypersensitivity at H3 somatodendritic autoreceptors and at heteroreceptors attributable to an increased number of HA binding sites could account for the various changes observed in this study.     

Benzimidazole-substituted (3-phenoxypropyl)amines as histamine H3 receptor ligands

A series of non-imidazole histamine H(3) receptor antagonists based on the (3-phenoxypropyl)amine motif, which is a common pharmacophore for H(3) antagonists, has been identified. A preliminary SAR study around the amine moiety has identified 8a as a potent H(3) antagonist possessing a good pharmacokinetic profile in the rat.     

Potential utility of histamine H3 receptor antagonist pharmacophore in antipsychotics

Histamine H₃ receptor (H₃R) antagonists have some antipsychotic properties although the clear molecular mechanism is still unknown. As actually the most effective and less side effective antipsychotics are drugs with multiple targets we have designed typical and atypical neuroleptics with an additional histamine H₃ pharmacophore. The 4-(3-piperidinopropoxy)phenyl pharmacophore moiety has been linked to amitriptyline, maprotiline, chlorpromazine, chlorprothixene, fluphenazine, and clozapine. Amide, amine and ester elements have been used generally to maintain or slightly shift affinity at dopamine D₂-like receptors (D₂ and D₃), to decrease affinity at histamine H₁ receptors, and to obtain H₃R ligands with low nanomolar or subnanomolar affinity. Change of effects at D₁-like receptors (D₁ and D₅) were heterogeneous. With these newly profiled compounds different antipsychotic properties might be achieved.     

N-Alkenyl and cycloalkyl carbamates as dual acting histamine H3 and H4 receptor ligands

Previous studies have shown that several imidazole derivatives possess affinity to histamine H(3) and H(4) receptors. Continuing our study on structural requirements responsible for affinity and selectivity for H(3)/H(4) receptor subtypes, two series of 3-(1H-imidazol-4-yl)propyl carbamates were prepared: a series of unsaturated alkyl derivatives (1-9) and a series possessing a cycloalkyl group different distances to the carbamate moiety (10-13). The compounds were tested for their affinities at the human histamine H(3) receptor, stably expressed in CHO-K1 cells. Compounds 1, 2, 5-7, 10-13 were investigated for their affinities at the human histamine H(4) receptor co-expressed with Gα(i2) and Gβ(1)γ(2) subunits in Sf9 cells. To expand the pharmacological profile, compounds were further tested for their H(3) receptor antagonist activity on guinea pig ileum and in vivo after oral administration to mice. All tested compounds exhibited good affinity for the human histamine H(3) receptor with K(i) values in the range from 14 to 194nM. All compounds were active in vivo after peroral administration (p.o.) to Swiss mice, thus demonstrating their ability to cross the blood-brain barrier. The most potent H(3) receptor ligand of these series was compound 5, 3-(1H-imidazol-4-yl)propyl pent-4-enylcarbamate with the highest affinity (K(i)=14nM). Additionally, compound 3 showed remarkable central nervous system (CNS) H(3)R activity, increasing the N(τ)-methylhistamine levels in mice with an ED(50) value of 0.55mg/kg, p.o. evidencing therefore, a twofold increase of inverse agonist/antagonist potency compared to the reference inverse agonist/antagonist thioperamide. In this study, the imidazole propyloxy carbamate moiety was kept constant. The different lipophilic moieties connected to the carbamate functionality in the eastern part of the molecule had a range of influences on the human H(4) receptor affinity (154-1326nM).     

Discovery of substituted benzyl tetrazoles as histamine H3 receptor antagonists

A series of potent and subtype selective H3 receptor antagonists containing a novel tetrazole core and diamine motif is reported. A one-pot multi-component Ugi reaction was utilised to rapidly develop the structure–activity relationships (SAR) of these compounds. Optimisation for liver microsome stability (t_1/2 >60 min), minimal CYP inhibition (IC₅₀ >50 μM) and high cell permeability (Caco-2 P_app >20 × 10^?6 cm/s) identified several compounds with drug-like properties.     

Validation of a histamine H3 receptor model through structure-activity relationships for classical H3 antagonists

Histamine H(3) receptor is a G protein-coupled receptor whose activation inhibits the synthesis and release of histamine and other neurotransmitters from nerve endings and is involved in the modulation of different central nervous system functions. H(3) antagonists have been proposed for their potential usefulness in diseases characterized by impaired neurotransmission and they have demonstrated beneficial effects on learning and food intake in animal models. In the present work, a 3D model of the rat histamine H(3) receptor, built by comparative modeling from the crystallographic coordinates of bovine rhodopsin, is presented with the discussion of its ability to predict the potency of known and new H(3) antagonists. A putative binding site for classical, imidazole-derived H(3) antagonists was identified by molecular docking. Comparison with a known pharmacophore model and the binding affinity of a new rigid H(3) antagonist (compound 1, pK(i)=8.02) allowed the characterization of a binding scheme which could also account for the different affinities observed in a recently reported series of potent H(3) antagonists, characterized by a 2-aminobenzimidazole moiety. Molecular dynamics simulations were employed to assess the stability and reliability of the proposed binding mode. Two new conformationally constrained benzimidazole derivatives were prepared and their binding affinity was tested on rat brain membranes; compound 9, designed to reproduce the conformation of a known potent H(3) antagonist, showed higher potency than compound 8, as expected from the binding scheme hypothesized.     

CLIC4 interacts with histamine H3 receptor and enhances the receptor cell surface expression

Histamine H3 receptor (H3R), one of G protein-coupled receptors (GPCRs), has been known to regulate neurotransmitter release negatively in central and peripheral nervous systems. Recently, a variety of intracellular proteins have been identified to interact with carboxy (C)-termini of GPCRs, and control their intracellular trafficking and signal transduction efficiencies. Screening for such proteins that interact with the C-terminus of H3R resulted in identification of one of the chloride intracellular channel (CLIC) proteins, CLIC4. The association of CLIC4 with H3R was confirmed in in vitro pull-down assays, coimmunoprecipitation from rat brain lysate, and immunofluorescence microscopy of rat cerebellar neurons. The data from flowcytometric analysis, radioligand receptor binding assay, and cell-based ELISA indicated that CLIC4 enhanced cell surface expression of wild-type H3R, but not a mutant form of the receptor that failed to interact with CLIC4. These results indicate that, by binding to the C-terminus of H3R, CLIC4 plays a critical role in regulation of the receptor cell surface expression.