Bodilisant—A Novel Fluorescent,Highly Affine Histamine H3 Receptor Ligand

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New 1‐Benzyl‐4‐hydroxypiperidine Derivatives as Non‐imidazole Histamine H3 Receptor Antagonists

A series of 1‐benzyl‐4‐(3‐aminopropyloxy)piperidine and 1‐benzyl‐4‐(5‐aminopentyloxy)piperidine derivatives has been prepared. The 1‐benzyl‐4‐hydroxypiperidine derivatives obtained were evaluated for their affinities at recombinant human histamine H₃ receptor, stably expressed in HEK 293T cells. All compounds investigated show moderate to pronounced in‐vitro affinities. The most potent antagonists in this series 9b2 (hH₃R, pK_i = 7.09), 9b1 (hH₃R, pK_i = 6.78), 9b5 (hH₃R, pK_i = 6.99), and 9b6 (hH₃R, pK_i = 6.97) were also tested in vitro as H₃ receptor antagonists – the electrically evoked contraction of the guinea‐pig jejunum. The histaminergic H₁ antagonism of selected compounds 9b1 , 9b2 , and 9b4 – 9b6 was established on the isolated guinea‐pig ileum by conventional methods; the pA₂ values were compared with the potency of pyrilamine. The compounds did not show any H₁ antagonistic activity (pA₂ < 4; for pyrilamine pA₂ = 9.53).     

Histamine H4 receptor-RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H4 receptor ligands

The human histamine H₄ receptor (hH₄R), co-expressed with Gα_i2 and Gβ₁γ₂ in Sf9 cells, is highly constitutively active. In the steady-state GTPase assay, the full agonist histamine (HA) induces only a relatively small signal (∼20-30%), resulting in a low signal-to background ratio. In order to improve this system for ligand screening purposes, the effects of the regulators of G-protein signaling (RGS) RGS4 and RGS19 (GAIP) were investigated. RGS4 and GAIP were fused to the C-terminus of hH₄R or co-expressed with non-fused hH₄R, always combined with Gα_i2 and Gβ₁γ₂. The non-fused RGS proteins did not significantly increase the relative effect of HA. With the hH₄R-RGS4 fusion protein the absolute GTPase activities, but not the relative HA-induced signal were increased. Fusion of hH₄R with GAIP caused a selective increase of the HA signal, resulting in an enhanced signal-to-noise ratio. A detailed characterization of the hH₄R-GAIP fusion protein (co-expressed with Gα_i2 and Gβ₁γ₂) and a comparison with the data obtained for the non-fused hH₄R (co-expressed with Gα_i2 and Gβ₁γ₂) led to the following results: (i) the relative agonist- and inverse agonist-induced signals at hH₄R-GAIP are markedly increased. (ii) Compared to the wild-type hH₄R, standard ligands show unaltered potencies and efficacies at hH₄R-GAIP. (iii) Like hH₄R, hH₄R-GAIP shows high and NaCl-resistant constitutive activity. (iv) hH₄R-GAIP shows the same G-protein selectivity profile as the non-fused hH₄R. Collectively, hH₄R-GAIP provides a sensitive test system for the characterization of hH₄R ligands and can replace the non-fused hH₄R in steady-state GTPase assays.     

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants

Background and Purpose

Some histamine H₄ receptor ligands act as inverse agonists at the human H₄ receptor (hH₄R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H₄ receptor (mH₄R) and rat H₄ receptor (rH₄R). To study molecular determinants of constitutive activity, H₄ receptor reciprocal mutants were constructed: single mutants: hH₄R-F169V, mH₄R-V171F, hH₄R-S179A, hH₄R-S179M; double mutants: hH₄R-F169V+S179A, hH₄R-F169V+S179M and mH₄R-V171F+M181S.

Experimental Approach

Site-directed mutagenesis with pVL1392 plasmids containing hH₄ or mH₄ receptors were performed. Wild-type or mutant receptors were co-expressed with Gα_i2 and Gβ₁γ₂ in Sf9 cells. Membranes were studied in saturation and competition binding assays ([³H]-histamine), and in functional [³⁵S]-GTPγS assays with inverse, partial and full agonists of the hH₄ receptor.

Key Results

Constitutive activity decreased from the hH₄ receptor via the hH₄R-F169V mutant to the hH₄R-F169V+S179A and hH₄R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH₄ receptor. Partial inverse hH₄ receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH₄ receptor agonists showed decreased maximal effects and potencies at hH₄R-F169V and double mutants. However, the mutation of S179 in the hH₄ receptor to M as in mH₄ receptor or A as in rH₄ receptor did not significantly reduce constitutive activity.

Conclusions and Implications

F169 and S179 are key amino acids for the high constitutive activity of hH₄ receptors and may also be of relevance for other constitutively active GPCRs.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc     

Expression and functional properties of canine,rat, and murine histamine H<Subscript>4</Subscript> receptors in Sf9 insect cells

The histamine H₄ receptor (H₄R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H₄R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H₄R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H₄R in comparison to human H₄R expressed in Sf9 insect cells. Only hH₄R and cH₄R exhibited a sufficiently high [³H]histamine affinity for radioligand binding studies. Generally, cH₄R exhibited lower ligand-affinities than hH₄R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH₄R than at other H₄R species orthologs. Unlike the other H₄R species orthologs, hH₄R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H₄R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH₄R, rH₄R, and mH₄R, whereas at hH₄R, JNJ7777120 was a partial inverse agonist. H₄R agonists from the class of N ^G -acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H₄R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H₄R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H₄R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of “the prototypical H₄R antagonist” JNJ7777120 as H₄R antagonism.     

Role of the second and third extracellular loops of the histamine H<Subscript>4</Subscript> receptor in receptor activation

The histamine H₄ receptor subtype (H₄R) belongs to the class 1 of G protein-coupled receptors and is involved in inflammatory and immunological processes. The aim of this study was to elucidate the importance of extracellular regions for the large species differences between human (h) and canine (c) H₄R. Therefore, chimeric receptors were generated by replacing corresponding domains of the hH₄R with canine N-terminus (h_cNTH₄R) and three canine extracellular loops, respectively (h_cE1H₄R, h_cE2H₄R and h_cE3H₄R). Wild type and chimeric H₄ receptors were expressed in Sf9 insect cells and subsequently characterized in [³H]histamine-binding experiments and in steady-state GTPase activity assays, where standard H₄R ligands histamine, 5-methylhistamine, thioperamide, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) and clozapine were examined. The exchange of N-terminus or first extracellular loop did not influence hH₄R pharmacology. The effect of altered second extracellular loop (E2-loop) and third extracellular loop (E3-loop) was rather ligand specific than agonist/inverse agonist specific. At h_cE3H₄R, the potency of histamine and 5-methylhistamine significantly decreased. The efficacy of the inverse agonist thioperamide was strongly reduced at h_cE2H₄R and h_cE3H₄R. Surprisingly, JNJ7777120 as weak inverse agonist at hH₄R exhibited partial agonistic activity at h_cE2H₄R and h_cE3H₄R. Molecular dynamic simulations suggest that the E2- and E3-loops are independently of each other involved in the partial/inverse agonism of JNJ7777120 and that E2- as well as E3-loop do not directly interact with JNJ7777120 in the binding pocket. In conclusion, our study indicates an involvement of the E2- and E3-loops in H₄R activation process after binding of some but not all examined ligands.     

Pharmacological and SAR analysis of the LINS01 compounds at the human histamine H1, H2, and H3 receptors

Histamine is a transmitter that activates the four receptors H₁R to H₄R. The H₃R is found in the nervous system as an autoreceptor and heteroreceptor, and controls the release of neurotransmitters, making it a potential drug target for neuropsychiatric conditions. We have previously reported that the 1‐(2,3‐dihydro‐1‐benzofuran‐2‐yl)methylpiperazines (LINS01 compounds) have the selectivity for the H₃R over the H₄R. Here, we describe their pharmacological properties at the human H₁R and H₂R in parallel with the H₃R, thus providing a full analysis of these compounds as histamine receptor ligands through reporter gene assays. Eight of the nine LINS01 compounds inhibited H₃R‐induced histamine responses, but no inhibition of H₂R‐induced responses was seen. Three compounds were weakly able to inhibit H₁R‐induced responses. No agonist responses were seen to any of the compounds at any receptor. SAR analysis shows that the N‐methyl group improves H₃R affinity while the N‐phenyl group is detrimental. The methoxy derivative, LINS01009, had the highest affinity.     

1‐[(2,3‐Dihydro‐1‐benzofuran‐2‐yl) methyl]piperazines as novel anti‐inflammatory compounds: Synthesis and evaluation on H3R/H4R

The histamine receptors (HRs) are members of G‐protein‐coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H₃R and H₄R have been explored as targets for drug discovery, including in the search for dual‐acting H₃R/H₄R ligands. The H₄R, the most recent histamine receptor, is a promising target for novel anti‐inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1‐[(2,3‐dihydro‐1‐benzofuran‐2‐yl)methyl]piperazines were synthesized and evaluated in competitive binding assays as H₃R/H₄R ligands herein. The results showed the compounds presented affinity (K_i) for H₃R/H₄R in micromolar range, and they are more selective to H₃R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl‐substituted compound LINS01005 has shown the higher affinity of the set for H₄R, but no considerable selectivity toward this receptor over H₃R. LINS01005 showed interesting anti‐inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX‐2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti‐inflammatory agents.     

Comparison of the pharmacological properties of human and rat histamine H3-receptors

Ligand pharmacology of histamine H₃-receptors is species-dependent. In previous studies, two amino acids in transmembrane domain 3 (TM III) were shown to play a significant role. In this study, we characterized human and rat histamine H₃-receptors (hH₃R and rH₃R, respectively), co-expressed with mammalian G proteins in Sf9 insect cell membranes. We compared a series of imidazole-containing H₃R ligands in radioligand binding and steady-state GTPase assays. H₃Rs similarly coupled to Gα_i/o-proteins. Affinities and potencies of the agonists histamine, N^α-methylhistamine and R-(α)-methylhistamine were in the same range. Imetit was only a partial agonist. The pharmacology of imetit and proxifan was similar at both species. However, impentamine was more potent and efficacious at rH₃R. The inverse agonists ciproxifan and thioperamide showed higher potency but lower efficacy at rH₃R. Clobenpropit was not species-selective. Strikingly, imoproxifan was almost full agonist at hH₃R, but an inverse agonist at rH₃R. Imoproxifan was docked into the binding pocket of inactive and active hH₃R- and rH₃R-models and molecular dynamic simulations were performed. Imoproxifan bound to hH₃R and rH₃R in E-configuration, which represents the trans-isomer of the oxime-moiety as determined in crystallization studies, and stabilized active hH₃R-, but inactive rH₃R-conformations. Large differences in electrostatic surfaces between TM III and TM V cause differential orientation of the oxime-moiety of imoproxifan, which then differently interacts with the rotamer toggle switch Trp^6.48 in TM VI. Collectively, the substantial species differences at H₃Rs are explained at a molecular level by the use of novel H₃R active-state models.     

Detailed analysis of biased histamine H4 receptor signalling by JNJ 7777120 analogues

Background and Purpose

The histamine H₄ receptor, originally thought to signal merely through Gα_i proteins, has recently been shown to also recruit and signal via β-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in β-arrestin2 recruitment, we have identified additional biased hH₄R ligands that preferentially couple to Gα_i or β-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH₄R signalling.

Experimental Approach

We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH₄R-mediated Gα_i protein signalling or β-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure–activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH₄R homology model was used to identify receptor regions important for biased hH₄R signalling.

Key Results

One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gα_i and β-arrestin2 pathway and was classified as unbiased hH₄R ligand. The other 47 indolecarboxamides were β-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as β-arrestin2-biased indolecarboxamides to induce β-arrestin2 recruitment could be correlated with different ligand features and hH₄R regions.

Conclusion and Implications

Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and β-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-β-arrestin2 recruitment. This knowledge is useful for the design of hH₄R ligands with biased intrinsic activities and aids our understanding of the mechanism of H₄R activation.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

Bronchospasmolytic activity and adenosine receptor binding of some newer 1,3‐dipropyl‐8‐phenyl substituted xanthine derivatives

The aldehyde derivatives of 1,3‐dipropyl xanthines as described in this paper, constitutes a new series of selective adenosine ligands displaying bronchospasmolytic activity. The effect of substitution at third‐ and fourth‐position of 8‐phenyl xanthine has also been taken into consideration. The synthesized compounds showed varying binding affinities at different adenosine receptor subtypes (A₁, A_2A, A_2B, and A₃) and also good in vivo bronchospasmolytic activity against histamine aerosol‐induced asthma in guinea pigs. Most of the compounds showed maximum affinity toward the A_2A receptor subtype. The monosubstituted 3‐aminoalkoxyl 8‐phenyl xanthine with a aminodiethyl moiety (compound 12e ) was found to be most potent A_2Aadenosine receptor ligand (K_i = 0.036 µM) followed by disubstituted 4‐aminoalkoxyl‐3‐methoxy‐8‐phenyl xanthine (K_i = 0.050 µM) (compound 10a ).     

Dual-acting diether derivatives of piperidine and homopiperidine with histamine H(3) receptor antagonistic and anticholinesterase activity

The study presents novel biological properties of diether derivatives of homo‐ or substituted piperidine ligands of the histamine H₃ receptor. The compounds were evaluated for their inhibitory potency against acetylcholinesterase (AChE) from the electric eel and butyrylcholinesterase (BuChE) from horse serum. The most interesting multifunctional compound 13 displayed high affinity for the cloned hH₃R (K_i = 3.48 nM) and moderate inhibitory potency against both enzymes (IC₅₀ AChE = 7.91 µM and BuChE = 4.97 µM). Molecular modeling studies revealed interactions with key amino acid residues in the homology model of histamine H₃ receptor ligands, as well as the binding model for AChE and BuChE in the catalytic and peripheral active sites.     

The Synthesis of 1,3,5‐triazine Derivatives and JNJ7777120 Analogues with Histamine H4 Receptor Affinity and Their Interaction with PTEN Promoter

The involvement of histamine and H₄ receptor (H₄R) in cancer has been investigated recently using the H₄R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a group of compounds with histamine H₄ receptor (H₄R) affinity on the promoter of PTEN gene encoding the antitumor PTEN protein. The series of novel compounds based either on H₄R antagonists JNJ7777120 structure or 1,3,5‐triazine scaffold were synthesized, evaluated for histamine H₄R affinity and used in this study. Compounds 5 and 7 belonging to the group of JNJ7777120 analogues showed the highest interaction with the promoter of PTEN gene and weak affinity against H₄R with K_i value >100 μm . These compounds showed no significant effect on neuroblastoma IMR‐32 cells viability indicating no correlation between PTEN gene promoter affinity and antitumor activity. Compound 6 , another JNJ7777120 analogue, showed the highest effect on IMR‐32 viability with calculated IC₅₀ = 23.27 μm . The 1,3,5‐triazine derivatives exhibited generally low or medium interaction with PTEN gene promoter. However, the 1,3,5‐triazine derivative 11 with the para‐bromo substituent showed the highest affinity against H₄R with K_i value of 520 nm and may be considered as a new lead structure.     

Canavanine increases glucose uptake in C2C12 cells through the activation of imidazoline I‐2B receptors

Canavanine is a guanidinium derivative that contains the basic structure of the ligand(s) of imidazoline receptor (I‐R). Canavanine has been reported to activate the imidazoline I‐3 receptor (I‐3R) both in vivo and in vitro. Additionally, the activation of the imidazoline I‐2B receptor (I‐2BR) by guanidinium derivatives may increase glucose uptake. Therefore, the effect of canavanine on the I‐2BR was investigated in the present study. Glucose uptake into cultured C₂C₁₂ cells was determined using the radio‐ligated tracer 2‐[¹⁴C]‐deoxy‐glucose. The changes in 5′ AMP‐activated protein kinase (AMPK) expression were also identified using Western blotting analysis. The canavanine‐induced glucose uptake was inhibited in a dose‐dependent manner by BU224 (0.01–1 μmol/L), which is a specific I‐2BR antagonist, in the C₂C₁₂ cells. Additionally, the canavanine‐stimulated AMPK phosphorylation and glucose transporter (GLUT4) expression were also sensitive to BU224 inhibition in the C₂C₁₂ cells. Moreover, both canavanine‐stimulated glucose uptake and AMPK phosphorylation were attenuated by high concentrations of amiloride (1–2 μmol/L), which is another established I‐2BR inhibitor, in a dose‐dependent manner in C₂C₁₂ cells. Additionally, compound C abolished the canavanine‐induced glucose uptake and AMPK phosphorylation at a concentration (0.1 μmol/L) sufficient to inhibit AMPK. In conclusion, these data demonstrated that canavanine has an ability to activate I‐2BR through the AMPK pathway to increase glucose uptake, which indicates I‐2BR as a new target for diabetic therapy.     

Influence of the N-terminus and the E2-loop onto the binding kinetics of the antagonist mepyramine and the partial agonist phenoprodifen to H(1)R

Numerous competitive radioligand binding studies revealed significant differences between human and guinea pig histamine H₁-receptors (hH₁R and gpH₁R), e.g. for the partial H₁R agonist phenoprodifen. But until now, there are only few studies with regard to binding kinetics at H₁R. Previous studies from our group revealed an influence of the exchange of N-terminus and E2-loop between hH₁R and gpH₁R onto affinity of phenoprodifen to H₁R (Strasser A, Wittmann HJ, Seifert R, J Pharmacol Exp Ther 326:783–791, 2008). The aim of this study was, therefore, to examine the impact of the N-terminus and the E2-loop on binding kinetics of the H₁R. The wild type hH₁R and gpH₁R and the chimeric h_gpE2H₁R (E2-loogp from guinea pig) and h_gpNgpE2H₁R (N-terminus and E2-loop from guinea pig) were co-expressed with regulator of G-protein signaling protein RGS4 in Sf9 insect cells and kinetic binding studies were performed using the antagonist [³H]mepyramine as radioligand. The rate constants for association and dissociation were, in dependence of the ligand, different between hH₁R and gpH₁R. Furthermore, the rate constants for association at h_gpNgpE2H₁R were significantly different compared to hH₁R and gpH₁R. Molecular dynamic simulation studies detected different interactions of amino acid side chains on the extracellular surface of the receptor. Based on these findings, the influence of extracellular surface onto binding kinetics and binding affinity can be explained. Thus, the extracellular surface of G protein-coupled receptors for biogenic amines, exhibits influence onto kinetics of ligand binding, onto ligand recognition and ligand guiding into the binding pocket.     

Striking differences of action of lisuride stereoisomers at histamine H1 receptors

This study has characterised the pharmacological profile of some dopaminergic agents of the ergoline family including the antiparkinsonian drug 8S-lisuride at native guinea pig histamine H₁ receptors and recombinant human and guinea pig H₁ receptors. We used segments of guinea pig ileum to study contractile responses, Sf9 insect cell membranes expressing the guinea pig H₁ receptor (gpH₁R) and the human H₁ receptor (hH₁R) to analyse GTPase activity of G_q-proteins and we conducted [³H]mepyramine binding studies using recombinant gpH₁Rs and hH₁Rs. 8S-Lisuride acted as a potent partial agonist at H₁Rs of guinea pig ileum (pD₂ 7.6, E _max 28% of histamine-induced maximum response) and as a silent antagonist at recombinant gpH₁Rs (pA₂ 7.5) and hH₁Rs (pA₂ 7.7) in GTPase studies. In contrast, its epimeric counterpart, 8R-lisuride, lacked efficacy and showed much lower affinity for H₁Rs of both species than 8S-lisuride. High affinity of 8S-lisuride and low affinity of 8R-lisuride was also estimated for gpH₁Rs and hH₁Rs in radioligand binding studies. The 1-allylated derivative of 8S-lisuride, 1-allyl-8S-lisuride, was equipotent with its parent compound (pD₂ 7.7) and showed enhanced efficacy in guinea pig ileum and at recombinant gpH₁Rs in GTPase studies (E _max 53%, 32%). Other antiparkinsionian drugs such as 8S-terguride, pergolide, cabergoline and bromocriptine displayed lower affinities for H₁Rs than 8S-lisuride. In conclusion, our results show that the antiparkinsonian drug 8S-lisuride is dramatically more potent than its epimeric counterpart 8R-lisuride in all assays used. 8S-Lisuride behaved as a partial agonist at gpH₁Rs and as a silent antagonist at hH₁Rs. Thus 8S-lisuride may act as an antagonist in vivo. This may be of potential importance since H₁Rs modulate dopaminergic transmission in the brain.     

Pseudopeptides containing the 2‐hydrazonoacyl fragment: analogs of the chemotactic agent HCO‐Met‐Leu‐Phe‐OMe

Abstract: In order to further examine the properties of pseudopeptides containing the 2‐hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N‐formyl‐tripeptide HCO‐Met‐Leu‐Phe‐OMe were designed and synthesized. The first group contains the new fragment as the N‐terminal residue and is represented by the N‐aryl derivatives p‐Cl‐C₆H₄‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 2 and 3 ) and by the corresponding N‐aroyl analogs p‐CH₃‐C₆H₄‐CO‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 4 ). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO‐Xaa‐NH‐N=C(R)‐CO‐Phe‐OMe ( 7a and 7b ) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.     

Receptor-specific functional efficacies of alkyl imidazoles as dual histamine H3/H4 receptor ligands

Histamine H₃ and H₄ receptors are highly related G protein-coupled receptors. Preclinical and clinical data strongly suggest the potential therapeutic application of selectively acting histamine H₄ receptor ligands to inflammatory conditions but also hint at a certain interference of the two receptors in diseases attended with itch and pain. The aim of this investigation was to identify dual acting ligands as pharmacological tools. Receptor binding profiles of ω-(1H-imidazol-4-yl)alkyl derivatives structurally defined as amides, carbamates, esters, ethers, ketones and ureas were evaluated with respect to their potencies at histamine H₃ and H₄ receptors. A two-step screening method based on in vitro radioligand binding studies and functional [³⁵S]GTPγS binding experiments was performed. The examined series of imidazole-containing compounds displayed both, selective histamine H₄ receptor and dual acting histamine H₃/H₄ receptor ligands. Slight structural modifications caused major differences in selectivity profiles and on functional properties at the human histamine H₄ receptor. N-(3-(1H-Imidazol-4-yl)propyl)-2-cyclohexylacetamide 11 was identified as most potent and selective human histamine H₄ receptor ligand in this series (K_i = 45 nM). Amide- and ether-containing structures consistently exhibited partial agonist efficacies, whereas ureas, ketones, esters and carbamates mainly acted as antagonists and inverse agonists. We identified novel dual acting histamine H₃/H₄ receptor ligands with varying efficacies at the histamine H₄ receptor subtype, whereas histamine H₃ receptor antagonism was kept constant, e.g. 3-(1H-imidazol-4-yl)propyl (cyclohexylmethyl)carbamate 19 or 4-(3-(3-phenylpropylthio)propyl)-1H-imidazole 30. These compounds state valuable pharmacological tools in studies of diseases, in which histamine H₃ and H₄ receptor signalling contributes to pathophysiological conditions.     

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 983.36 of the human histamine H4 receptor

Background and Purpose

The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H₄ receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182^5.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98^3.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98^3.36 by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H₄ receptor research.

Experimental Approach

We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH₄ receptor radioligand binding, G protein activation and β-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically.

Key Results

VUF14480 was shown to be a partial agonist of hH₄ receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH₄ receptor with submicromolar affinity. Serine substitution of C98^3.36 prevented this covalent interaction.

Conclusion and Implications

VUF14480 is thought to bind covalently to the hH₄ receptor-C98^3.36 residue and partially induce hH₄ receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH₄ receptor.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

Synthesis,Antibacterial Activities,and 3D‐QSAR of Sulfone Derivatives Containing 1, 3, 4‐Oxadiazole Moiety

A series of sulfone derivatives containing 1, 3, 4‐oxadiazole moiety were prepared and evaluated for their antibacterial activities by the turbidimeter test. Most compounds inhibited growth of Ralstonia solanacearum (R. solanacearum) from tomato and tobacco bacterial wilt with high potency, among which compounds 5a and 5b exhibited the most potent inhibition against R. solanacearum from tomato and tobacco bacterial wilts with EC₅₀ values of 19.77 and 8.29 μg/mL, respectively. Our results also demonstrated that 5a, 5b , and a number of other compounds were more potent than commercial bactericides Kocide 3000 and Thiodiazole Copper, which inhibited R. solanacearum from tomato bacterial wilt with EC₅₀ values of 93.59 and 99.80 μg/mL and tobacco bacterial wilt with EC₅₀ values of 45.91 and 216.70 μg/mL, respectively. The structure–activity relationship (SAR) of compounds was studied using three‐dimensional quantitative structure–activity relationship (3D‐QSAR) models created by comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) based on compound bioactivities against tomato and tobacco bacterial wilts. The 3D‐QSAR models effectively predicted the correlation between inhibitory activity and steric–electrostatic properties of compounds.