Molecular Modeling of Histamine Receptors-Recent Advances in Drug Discovery.

The recent developments of fast reliable docking, virtual screening and other algorithms gave rise to discovery of many novel ligands of histamine receptors that could be used for treatment of allergic inflammatory disorders, central nervous system pathologies, pain, cancer and obesity. Furthermore, the pharmacological profiles of ligands clearly indicate that these receptors may be considered as targets not only for selective but also for multi-target drugs that could be used for treatment of complex disorders such as Alzheimer's disease. Therefore, analysis of protein-ligand recognition in the binding site of histamine receptors and also other molecular targets has become a valuable tool in drug design toolkit. This review covers the period 2014-2020 in the field of theoretical investigations of histamine receptors mostly based on molecular modeling as well as the experimental characterization of novel ligands of these receptors.

Nootkatone Inhibits Acute and Chronic Inflammatory Responses in Mice.

Nootkatone (NTK) is a sesquiterpenoid found in essential oils of many species of Citrus (Rutaceae). Considering previous reports demonstrating that NTK inhibited inflammatory signaling pathways, this study aimed to investigate the effects of this compound in mice models of acute and chronic inflammation. Murine models of paw edema induced by carrageenan, dextran, histamine, and arachidonic acid, as well as carrageenan-induced peritonitis and pleurisy, were used to evaluate the effects of NTK on acute inflammation. A murine model of granuloma induced by cotton pellets was used to access the impact of NTK treatment on chronic inflammation. In the acute inflammation models, NTK demonstrated antiedematogenic effects and inhibited leukocyte recruitment, which was associated with decreased vascular permeability, inhibition of myeloperoxidase (MPO), interleukin (IL)1-ß, and tumor necrosis factor (TNF)-α production. In silico analysis suggest that NTZ anti-inflammatory effects may also occur due to inhibition of cyclooxygenase (COX)-2 activity and antagonism of the histamine receptor type 1 (H1). These mechanisms might have contributed to the reduction of granuloma weight and protein concentration in the homogenates, observed in the chronic inflammation model. In conclusion, NTK exerted anti-inflammatory effects that are associated with inhibition of IL1-ß and TNF-α production, possibly due to inhibition of COX-2 activity and antagonism of the H1 receptor. However, further studies are required to characterize the effects of this compound on chronic inflammation.

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

Research Advances and Prospects on Mechanism of Sinomenin on Histamine Release and the Binding to Histamine Receptors.

Sinomenine (SIN) is widely used in China to treat a variety of rheumatic diseases (RA), and has various pharmacological effects such as anti-inflammatory, analgesic, and anti-tumor effects. However, due to the histamine release characteristics of SIN, its adverse reactions such as allergic reactions, gastrointestinal reactions, and circulatory systemic reactions have been drawing increasing attention. We present here a systematic review of the chemical structure, pharmacological effects, clinical application, and adverse reactions of SIN, a detailed discussion on the relationship between histamine/histamine receptor and mechanism of action of SIN. In addition, we simulated the binding of SIN to four histamine receptors by using a virtual molecular docking method and found that the bonding intensity between SIN and receptors varied in the order shown as follows: H1R > H2R ~ H3R > H4R. The docking results suggested that SIN might exhibit dual regulatory effects in many processes such as cyclooxygenase-2 (COX-2) expression, NF-κB pathway activation, and degranulation of mast cells to release histamine, thereby exhibiting pro-inflammatory (adverse reactions)/anti-inflammatory effects. This study provides a theoretical basis for the clinical treatment of inflammations seen such as in RA using SIN, and also suggests that SIN has great potential in the field of cancer treatment and will have very important social and economic significance.

Sodium binding to hH3R and hH 4R--a molecular modeling study.

Several aminergic GPCRs, e.g., the human histamine H3-receptor (hH3R) are sensitive to sodium ions. Based on these experimental results, including site directed mutagenesis studies, a sodium binding pocket near to the highly conserved Asp2.50 was suggested. Recently, in the crystallized adenosine A2A receptor (4EIY), a sodium ion was found in a pocket, coordinated by Asp52, Ser91, and three water molecules. Despite high homology in amino acid sequence between hH3R and hH4R, pharmacological studies revealed that the hH4R is--in contrast to hH3R--not sensitive to sodium ions. In order to obtain a deeper insight onto the differences in sodium sensitivity between hH3R and hH4R, we performed molecular modelling studies, including molecular dynamic simulations and calculation of Gibbs energy of solvation. The results of the modeling studies suggested that the amino acid at position 7.42 influences sodium binding to aminergic GPCRs in different ways. A comparison of the amino acids forming the sodium binding channel between the ligand binding pocket and the sodium binding pocket of all human aminergic GPCRs showed an 80% occurrence of glycine--in contrast to hH3R and hH4R. The Gln7.42 at hH4R disrupts a water chain, connecting the Asp3.32 of the orthosteric binding site and the Asp2.50 of the allosteric binding site. Besides, the oxygen of the glutamine side chain stabilizes the interaction of the sodium ion with the Asp3.32. Thus, the binding of the sodium into the allosteric binding site might be hindered kinetically.

Aryl-1,3,5-triazine derivatives as histamine H4 receptor ligands.

A series of novel 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazine derivatives with different aryl substituents in the 6-position was designed, synthesized and evaluated for histamine H4 receptor (H4R) affinity in Sf9 cells expressing human H4R co-expressed with G-protein subunits. Triazine derivative 8 with a 6-(p-chlorophenyl) substituent showed the highest affinity with hH4R Ki value of 203 nM and was classified as an antagonist in cAMP accumulation assay. This compound, identified as a new lead structure, demonstrated also anti-inflammatory properties in preliminary studies in mice (carrageenan-induced edema test) and neither possessed significant antiproliferative activity, nor modulated CYP3A4 activity up to concentration of 25 µM. In order to discuss structure-activity relationships molecular modeling and docking studies were undertaken.

Discovery of novel lead in the group of N-substituted piperazine ether derivatives with potential histamine H3 receptor activity.

The search for novel lead from the group of various substituted N-piperazine ether derivatives was performed. Acyl- and pyridylpiperazine ethyl/propyl ethers were obtained via three different synthetic pathways. Affinity to histamine H3 receptor was established, as well as, for selected compounds, selectivity towards histamine H4R. Docking studies to the histamine H3R homology model strengthened the position of (4-(3-(4-(3-chlorobenzoyl)piperazin-1- yl)propoxy)phenyl)(cyclopropyl)methanone (compound 26) as a novel lead for further studies on histamine H3 receptor antagonist/inverse agonist.

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) 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 ß-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 ß-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 ß-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.

Use of PD biomarkers to drive dose selection and early clinical decision making.

A major challenge facing the development of new therapies is the high level of compound attrition in late-stage clinical studies. A key factor in reducing these unsustainable levels of attrition is the successful evaluation of the level of drug effect on its target pathway in early development, otherwise known as testing the compound mechanism. Incorporation of PD biomarkers into Phase I/II trials to demonstrate compound binding to its molecular target and the subsequent modulation of downstream pathways enables early testing of compound mechanism and provides a data-driven framework for decisions on compound progression. This review will discuss the identification and validation of such 'fit-for-purpose' PD biomarkers, and case studies illustrating their use and value in dose selection and accelerating the clinical development of small-molecule drugs will be described.

Molecular pharmacology of histamine H4 receptors.

The histamine H4 receptor (H4R) is the youngest member of the histamine receptor family. Based on its predominant expression pattern in hematopoietic cells, the H4R is considered to be an interesting drug target for inflammatory disorders such as allergy and asthma. Since the identification and cloning of the H4R in 2000, drug discovery programs boosted the development of various H4R (specific) ligands. Differences between H4R orthologs in combination with available three-dimensional G protein-coupled receptor (GPCR) models have guided site-directed mutagenesis studies to gain insight in ligand binding and receptor activation. In addition, ongoing characterization of H4R-mediated signaling in transfected and native cells contributes to further unravel the (patho-) physiological functions of H4Rs.

High-resolution metabolic profiling towards G protein-coupled receptors: rapid and comprehensive screening of histamine H4 receptor ligands.

In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H(4) receptor (hH(4)R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH(4)R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic 'hotspots' for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor.

The H(4)R (histamine H(4) 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 H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.

Discovery of novel human histamine H4 receptor ligands by large-scale structure-based virtual screening.

A structure-based virtual screening (SBVS) was conducted on a ligand-supported homology model of the human histamine H4 receptor (hH4R). More than 8.7 million 3D structures derived from different vendor databases were investigated by docking to the hH4R binding site using FlexX. A total of 255 selected compounds were tested by radioligand binding assay and 16 of them possessed significant [(3)H]histamine displacement. Several novel scaffolds were identified that can be used to develop selective H4 ligands in the future. As far as we know, this is the first SBVS reported on H4R, representing one of the largest virtual screens validated by the biological evaluation of the virtual hits.

Copper(II) interaction with prion peptide fragments encompassing histidine residues within and outside the octarepeat domain: speciation, stability constants and binding details.

A 31-mer polypeptide, which encompasses residues 84-114 of human prion protein HuPrP(84-114) and contains three histidyl residues, namely one from the octarepeat (His85) and two histidyl residues from outside the octarepeat region (His96 and His111), and its mutants with two histidyl residues HuPrP(84-114)His85Ala, HuPrP(84-114) His96Ala, HuPrP(84-114)His111Ala and HuPrP(91-115) have been synthesised and their Cu2+ complexes studied by potentiometric and spectroscopic (UV/Vis, CD, EPR, ESI-MS) techniques. The results revealed a high Cu2+-binding affinity of all peptides, and the spectroscopic studies made it possible to clarify the coordination mode of the peptides in the different complex species. The imidazole nitrogen donor atoms of histidyl residues are the exclusive metal-binding sites below pH 5.5, and they have a preference for macrochelate structure formation. The deprotonation and metal-ion coordination of amide functions take place by increasing the pH; all of the histidines can be considered to be independent metal-binding sites in these species. As a consequence, di- and trinuclear complexes can be present even in equimolar samples of the metal ion and peptides, but the ratios of polynuclear species do not exceed the statistically expected ones; this excludes the possibility of cooperative Cu2+ binding. The species with a (N(im),N,N)-binding mode are favoured around pH 7, and their stability is enhanced by the macrochelation from another histidyl residue in the mononuclear complexes. The independence of the histidyl sites results in the existence of coordination isomers and the preference for metal binding follows the order of: His111>His96>His85. Deprotonation and metal-ion coordination of the third amide functions were detected in slightly alkaline solutions at each of the metal-binding sites; all had a (N(im),N,N,N)-coordination mode. Spectroscopic measurements also made it clear that the four lysyl amino groups of the peptides are not metal-binding sites in any cases.

Molecular cloning and characterization of a new human histamine receptor, HH4R.

A new histamine receptor, HH4R, was cloned from human leukocyte cDNA. The deduced amino acid sequence showed about 40% identity to that of the human histamine H3 receptor, HH3R. HH4R-expressing cells responded to histamine, inhibiting forskolin-induced cAMP accumulation. An H3 agonist, N-alpha-methylhistamine (NAMHA), bound specifically to HH4R, while another H3 agonist, R(-)-alpha-methylhistamine (RAMHA), and the H3 antagonist, thioperamide, competed with this binding. RAMHA, NAMHA, and imetit inhibited forskolin-induced cAMP accumulation in HH4R-expressing cells. However, the binding affinities and agonistic activities of H3 agonists to HH4R were weaker than those to HH3R. Low expression of HH4R was detected in a wide variety of peripheral tissues by RT-PCR; however, in contrast with HH3R, expression was not detected in the brain. These observations indicate that the clone is a distinct histamine receptor from HH3R, and thus is named HH4R.

Tick histamine-binding proteins: isolation, cloning, and three-dimensional structure.

High-affinity histamine-binding proteins (HBPs) were discovered in the saliva of Rhipicephalus appendiculatus ticks. Their ability to outcompete histamine receptors indicates that they suppress inflammation during blood feeding. The crystal structure of a histamine-bound HBP, determined at 1.25 A resolution, reveals a lipocalin fold novel in containing two binding sites for the same ligand. The sites are orthogonally arranged and highly rigid and form an internal surface of unusual polar character that complements the physicochemical properties of histamine. As soluble receptors of histamine, HBPs offer a new strategy for controlling histamine-based diseases.

Histamine-induced Cl- secretion in human nasal epithelium: responses of apical and basolateral membranes.

The mechanism by which receptors coupled to phospholipase C (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (10(-4) M, basolateral administration) stimulated a rapid increase in equivalent short-circuit current, an index of Cl- secretion, that returned to baseline within 5 min. Intracellular recordings with double-barreled Cl(-)-selective microelectrodes showed that the apical and basolateral membrane potentials rapidly hyperpolarized, the fractional resistance of the apical membrane increased, and the transepithelial resistance decreased in response to histamine. Intracellular Cl- activity remained constant. Equivalent circuit analysis revealed that the early portion (< 0.9 min) of the Cl- secretory response was driven by an activation of a hyperpolarizing basolateral conductance, likely K+, whereas the later (> 0.9 min) phase of Cl- secretion reflects activation of the apical membrane Cl- conductance. Histamine raised intracellular Ca2+ (Ca2+i) measured by fura-2 in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasma membrane influx pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (15 microM), coupled with reduced bathing solution Ca2+, blunted the rise in Ca2+i and the net transepithelial Cl- secretory response to histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance, and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca2+i may participate in the activation of both the basolateral and apical conductances.