Insight into the Mode of Action of 8-Hydroxyquinoline-Based Blockers on the Histamine Receptor 2.

Histamine receptor 2 (HRH2) blockers are used to treat peptic ulcers and gastric reflux. Chlorquinaldol and chloroxine, which contain an 8-hydroxyquinoline (8HQ) core, have recently been identified as blocking HRH2. To gain insight into the mode of action of 8HQ-based blockers, here, we leverage an HRH2-based sensor in yeast to evaluate the role of key residues in the HRH2 active site on histamine and 8HQ-based blocker binding. We find that the HRH2 mutations D98A, F254A, Y182A, and Y250A render the receptor inactive in the presence of histamine, while HRH2:D186A and HRH2:T190A retain residual activity. Based on molecular docking studies, this outcome correlates with the ability of the pharmacologically relevant histamine tautomers to interact with D98 via the charged amine. Docking studies also suggest that, unlike established HRH2 blockers that interact with both ends of the HRH2 binding site, 8HQ-based blockers interact with only one end, either the end framed by D98/Y250 or T190/D186. Experimentally, we find that chlorquinaldol and chloroxine still inactivate HRH2:D186A by shifting their engagement from D98 to Y250 in the case of chlorquinaldol and D186 to Y182 in the case of chloroxine. Importantly, the tyrosine interactions are supported by the intramolecular hydrogen bonding of the 8HQ-based blockers. The insight gained in this work will aid in the development of improved HRH2 therapeutics. More generally, this work demonstrates that Gprotein-coupled receptor (GPCR)-based sensors in yeast can help elucidate the mode of action of novel ligands for GPCRs, a family of receptors that bind 30% of FDA therapeutics.

New Chemical Biology Tools for the Histamine Receptor Family.

The histamine research community has in the last decade been very active and generated a number of exciting new chemical biology tools for the study of histamine receptors, their ligands, and their pharmacology. In this paper we describe the development of histamine receptor structural biology, the use of receptor conformational biosensors, and the development of new ligands for covalent or fluorescent labeling or for photopharmacological approaches (photocaging and photoswitching). These new tools allow new approaches to study histamine receptors and hopefully will lead to better insights in the molecular aspects of histamine receptors and their ligands.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations.

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R-mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R-mGsi and -mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

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.

Cryo-EM structure of the human histamine H1 receptor/Gq complex.

Histamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of "squash to activate and expand to deactivate". The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-ß junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions.

G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.

Identification of antiviral antihistamines for COVID-19 repurposing.

There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with in vitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 in vitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes.

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

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.

Quantitative electrophysiological monitoring of anti-histamine drug effects on live cells via reusable sensor platforms.

We demonstrated the quantitative electrophysiological monitoring of histamine and anti-histamine drug effects on live cells via reusable sensor platforms based on carbon nanotube transistors. This method enabled us to monitor the real-time electrophysiological responses of a single HeLa cell to histamine with different concentrations. The measured electrophysiological responses were attributed to the activity of histamine type 1 receptors on a HeLa cell membrane by histamine. Furthermore, the effects of anti-histamine drugs such as cetirizine or chlorphenamine on the electrophysiological activities of HeLa cells were also evaluated quantitatively. Significantly, we utilized only a single device to monitor the responses of multiple HeLa cells to each drug, which allowed us to quantitatively analyze the antihistamine drug effects on live cells without errors from the device-to-device variation in device characteristics. Such quantitative evaluation capability of our method would promise versatile applications such as drug screening and nanoscale bio sensor researches.

Molecular Modelling Approaches for the Analysis of Histamine Receptors and Their Interaction with Ligands.

Several experimental techniques to analyse histamine receptors are available, e.g. pharmacological characterisation of known or new compounds by different types of assays or mutagenesis studies. To obtain insights into the histamine receptors on a molecular and structural level, crystal structures have to be determined and molecular modelling studies have to be performed. It is widely accepted to generate homology models of the receptor of interest based on an appropriate crystal structure as a template and to refine the resulting models by molecular dynamic simulations. A lot of modelling techniques, e.g. docking, QSAR or interaction fingerprint methods, are used to predict binding modes of ligands and pharmacological data, e.g. affinity or even efficacy. However, within the last years, molecular dynamic simulations got more and more important: First of all, molecular dynamic simulations are very helpful to refine the binding mode of a ligand to a histamine receptor, obtained by docking studies. Furthermore, with increasing computational performance it got possible to simulate complete binding pathways of ions or ligands from the aqueous extracellular phase into the allosteric or orthosteric binding pocket of histamine receptors.

Discovery of nanomolar ligands with novel scaffolds for the histamine H4 receptor by virtual screening.

The involvement of histamine H4 receptor (H4R) in immune cells chemotaxis and mediator release makes it an attractive target for the treatment of inflammation disorders. A decade of medicinal chemistry efforts has led to several promising ligands, although the chemical structures described so far possesses a singular limited diversity. We report here the discovery of novel structures, belonging to completely different scaffolds. The virtual screening was planed as a two-steps process. First, using a "scout screening" methodology, we have experimentally probed the H4R ligand binding site using a small size chemical library with very diverse structures, and identified a hit that further assist us in refining a raw 3D homology model. Second, the refined 3D model was used to conduct a widened virtual screening. This two-steps strategy proved to be very successful, both in terms of structural diversity and hit rate (23%). Moreover, the hits have high affinity for the H4R, with most potent ligands in the nanomolar range.

QSAR models on H4 receptor antagonists associated with inflammation and anaphylaxis.

Histamine, an endogenous amine is implicated in hypersensitivity (allergic) responses, gastric acid secretion, neurotransmission, immuno-modulation, cell differentiation, and embryonic development. It exerts its effects via four histamine receptor subtypes, termed H1 to H4 receptors (H1R-H4R) belonging to the superfamily of G-protein coupled receptors. The latest discovered histamine receptor, H4R, is implicated in the chemotaxis of several cell types and strongly associated with immune and inflammatory responses. Thus, we found interesting to analyze in terms of 2D-QSAR a number of H4 antagonists in order to highlight the most important physicochemical properties implicated in their mechanism of action and in continuation to suggest structural modifications. The C-QSAR platform of Biobyte has been used in this study. The study reveals that lipophilicity, clog P (linear or bilinear model) as well as steric factors such as the overall molar refractivity (CMR), molar volume or the substitutents molar refractivity (linear) or the sterimol parameters B1 and B5 are important. Electronic effects appear only in one model. The study shows that log P as calculated from the C-QSAR program of Biobyte is suitable for this form of QSAR study.

Structure-based discovery and binding site analysis of histamine receptor ligands.

INTRODUCTION: The application of structure-based drug discovery in histamine receptor projects was previously hampered by the lack of experimental structures. The publication of the first X-ray structure of the histamine H1 receptor has been followed by several successful virtual screens and binding site analysis studies of H1-antihistamines. This structure together with several other recently solved aminergic G-protein coupled receptors (GPCRs) enabled the development of more realistic homology models for H2, H3 and H4 receptors. Areas covered: In this paper, the authors review the development of histamine receptor models and their application in drug discovery. Expert opinion: In the authors' opinion, the application of atomistic histamine receptor models has played a significant role in understanding key ligand-receptor interactions as well as in the discovery of novel chemical starting points. The recently solved H1 receptor structure is a major milestone in structure-based drug discovery; however, our analysis also demonstrates that for building H3 and H4 receptor homology models, other GPCRs may be more suitable as templates. For these receptors, the authors envisage that the development of higher quality homology models will significantly contribute to the discovery and optimization of novel H3 and H4 ligands.

Conformational Restriction and Enantioseparation Increase Potency and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor Agonists.

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H4 receptor (hH4R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hHxR (x: 1-4) subtypes on Sf9 cell membranes (radioligand binding, [(35)S]GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H4R (HEK-293 cells). The most potent H4R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R, >100-fold selectivity; H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([(35)S]GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.

In silico modeling techniques for predicting the tertiary structure of human H4 receptor.

First cloned in 2000, the human Histamine H4 Receptor (hH4R) is the last member of the histamine receptors family discovered so far, it belongs to the GPCR super-family and is involved in a wide variety of immunological and inflammatory responses. Potential hH4R antagonists are proposed to have therapeutic potential for the treatment of allergies, inflammation, asthma and colitis. So far, no hH4R ligands have been successfully introduced to the pharmaceutical market, which creates a strong demand for new selective ligands to be developed. in silico techniques and structural based modeling are likely to facilitate the achievement of this goal. In this review paper we attempt to cover the fundamental concepts of hH4R structure modeling and its implementations in drug discovery and development, especially those that have been experimentally tested and to highlight some ideas that are currently being discussed on the dynamic nature of hH4R and GPCRs, in regards to computerized techniques for 3-D structure modeling.

Binding pathway of histamine to the hH4R, observed by unconstrained molecular dynamics.

Histamine binds with high affinity to the human histamine H4 receptor (hH4R). We are the first to examine the complete binding pathway of histamine from the extracellular side to the orthosteric binding site of the hH4R by means of unconstrained molecular dynamic simulation. Furthermore, the simulations show that the positively charged amine moiety of the histamine interacts electrostatically with the highly conserved Asp(3.32), while the imidazole moiety forms a hydrogen bond interaction with Glu(5.46) and Gln(7.42).

Sequential application of ligand and structure based modeling approaches to index chemicals for their hH4R antagonism.

The human histamine H4 receptor (hH4R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH4R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH4R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH4R bioactivity. An application of the prediction model on external test set composed of more than 160 hH4R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking ∼ 4000 chemicals highly indexed as H4R antagonists' candidates. Next, a series of 3D models of hH4R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH4R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.