Microliquid/Liquid Interfacial Sensors: Biomimetic Investigation of Transmembrane Mechanisms and Real-Time Determinations of Clemastine, Cyproheptadine, Epinastine, Cetirizine, and Desloratadine.

Antihistamines relieve allergic symptoms by inhibiting the action of histamine. Further understanding of antihistamine transmembrane mechanisms and optimizing the selectivity and real-time monitoring capabilities of drug sensors is necessary. In this study, a micrometer liquid/liquid (L/L) interfacial sensor has served as a biomimetic membrane to investigate the mechanism of interfacial transfer of five antihistamines, i.e., clemastine (CLE), cyproheptadine (CYP), epinastine (EPI), desloratadine (DSL), and cetirizine (CET), and realize the real-time determinations. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques have been used to uncover the electrochemical transfer behavior of the five antihistamines at the L/L interface. Additionally, finite element simulations (FEMs) have been employed to reveal the thermodynamics and kinetics of the process. Visualization of antihistamine partitioning in two phases at different pH values can be realized by ion partition diagrams (IPDs). The IPDs also reveal the transfer mechanism at the L/L interface and provide effective lipophilicity at different pH values. Real-time determinations of these antihistamines have been achieved through potentiostatic chronoamperometry (I-t), exhibiting good selectivity with the addition of nine common organic or inorganic compounds in living organisms and revealing the potential for in vivo pharmacokinetics. Besides providing a satisfactory surrogate for studying the transmembrane mechanism of antihistamines, this work also sheds light on micro- and nano L/L interfacial sensors for in vivo analysis of pharmacokinetics at a single-cell or single-organelle level.

Molecular mechanism of antihistamines recognition and regulation of the histamine H1 receptor.

Histamine receptors are a group of G protein-coupled receptors (GPCRs) that play important roles in various physiological and pathophysiological conditions. Antihistamines that target the histamine H1 receptor (H1R) have been widely used to relieve the symptoms of allergy and inflammation. Here, to uncover the details of the regulation of H1R by the known second-generation antihistamines, thereby providing clues for the rational design of newer antihistamines, we determine the cryo-EM structure of H1R in the apo form and bound to different antihistamines. In addition to the deep hydrophobic cavity, we identify a secondary ligand-binding site in H1R, which potentially may support the introduction of new derivative groups to generate newer antihistamines. Furthermore, these structures show that antihistamines exert inverse regulation by utilizing a shared phenyl group that inserts into the deep cavity and block the movement of the toggle switch residue W4286.48. Together, these results enrich our understanding of GPCR modulation and facilitate the structure-based design of novel antihistamines.

Cationic amphiphilic antihistamines inhibit STAT3 via Ca2+-dependent lysosomal H+ efflux.

Commonly used antihistamines and other cationic amphiphilic drugs (CADs) are emerging as putative cancer drugs. Their unique chemical structure enables CADs to accumulate rapidly inside lysosomes, where they increase lysosomal pH, alter lysosomal lipid metabolism, and eventually cause lysosomal membrane permeabilization. Here, we show that CAD-induced rapid elevation in lysosomal pH is caused by a lysosomal H+ efflux that requires P2RX4-mediated lysosomal Ca2+ release and precedes the lysosomal membrane permeabilization. The subsequent cytosolic acidification triggers the dephosphorylation, lysosomal translocation, and inactivation of the oncogenic signal transducer and activator of transcription 3 (STAT3) transcription factor. Moreover, CAD-induced lysosomal H+ efflux sensitizes cancer cells to apoptosis induced by STAT3 inhibition and acts synergistically with STAT3 inhibition in restricting the tumor growth of A549 non-small cell lung carcinoma xenografts. These findings identify lysosomal H+ efflux and STAT3 inhibition as anticancer mechanisms of CADs and reinforce the repurposing of safe and inexpensive CADs as cancer drugs with a drug combination strategy.

An In Vivo Definition of Brain Histamine Dynamics Reveals Critical Neuromodulatory Roles for This Elusive Messenger.

Histamine is well known for mediating peripheral inflammation; however, this amine is also found in high concentrations in the brain where its roles are much less known. In vivo chemical dynamics are difficult to measure, thus fundamental aspects of histamine's neurochemistry remain undefined. In this work, we undertake the first in-depth characterization of real time in vivo histamine dynamics using fast electrochemical tools. We find that histamine release is sensitive to pharmacological manipulation at the level of synthesis, packaging, autoreceptors and metabolism. We find two breakthrough aspects of histamine modulation. First, differences in H3 receptor regulation between sexes show that histamine release in female mice is much more tightly regulated than in male mice under H3 or inflammatory drug challenge. We hypothesize that this finding may contribute to hormone-mediated neuroprotection mechanisms in female mice. Second, a high dose of a commonly available antihistamine, the H1 receptor inverse agonist diphenhydramine, rapidly decreases serotonin levels. This finding highlights the sheer significance of pharmaceuticals on neuromodulation. Our study opens the path to better understanding and treating histamine related disorders of the brain (such as neuroinflammation), emphasizing that sex and modulation (of serotonin) are critical factors to consider when studying/designing new histamine targeting therapeutics.

Disruption of histamine/H1R-STAT3-SLC7A11 axis exacerbates doxorubicin-induced cardiac ferroptosis.

Doxorubicin (DOX) is widely used in the treatment of various cancers, increasing the great risk of adverse cardiovascular events, while the clinical intervention effect is not ideal. Histamine has been documented to participate in pathophysiological processes of cardiovascular diseases and inflammation-associated carcinogenesis. However, the potential roles of histamine in antitumor-related cardiotoxicity have not been fully elucidated. In this study, cardiomyocytes (hiPSC-CMs, HL-1 cells) and mice were treated with DOX to establish DOX-induced cardiotoxicity (DIC) models. Histidine decarboxylase knockout mice (HDC-/-) mice and histamine 1 receptor (H1R) antagonist were used to explore the effect of histamine/H1R signaling on DIC. Our results demonstrated that histamine deficiency or pharmaceutical inhibition of H1R accelerated myocardial ferroptosis, which is responsible for the aggravated DIC both in vivo and in vitro, while the supplementation of exogenous histamine reversed these changes. Our data revealed that the dysfunction of histamine/H1R signaling repressed the activation of transducer and activator of transcription 3 (STAT3), accompanying with decreased expression of solute carrier family7member11 (SLC7A11), a major modulator of ferroptosis. Conclusively, the disruption of histamine/H1R axis triggered ferroptosis and exacerbated DIC possibly by modulating STAT3-SLC7A11 pathway. Our findings point to a potential therapeutic target for DIC and provide more consideration on the usage of antihistamine drugs.

Repurposing of the antihistamine mebhydrolin napadisylate for treatment of Zika virus infection.

Zika virus (ZIKV) infection can lead to severe neurological disorders and fetal defects, which has become a public health problem worldwide. However, effective clinical treatment for ZIKV infection was still arduous. Antihistamines are attractive candidates for drug repurposing due to their low price and widespread availability. Here we screened FDA-approved antihistamine drugs to obtain anti-ZIKV candidate compounds and identified mebhydrolin napadisylate (MHL) that exhibits the potent inhibition of ZIKV infection in various cell lines in a histamine H1 receptor-independent manner. Mechanistic studies suggest that MHL acts as a ZIKV NS5 RNA-dependent RNA polymerase (RdRp) inhibitor, supported by a structure-activity relationship (SAR) analysis showing the correlation between the inhibitory effect upon viral RNA synthesis and ZIKV infectivity. Furthermore, MHL was shown to bind ZIKV NS5 RdRp in vitro and predicted to interact with key residues at the active site of ZIKV NS5 RdRp by molecular docking analysis. Our data together suggest that MHL suppresses ZIKV infection through the inhibition of ZIKV NS5 RdRp activity. This study highlights that MHL might be a promising clinical anti-ZIKV therapeutic.

Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical.

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Histamine H3 receptor and cholinesterases as synergistic targets for cognitive decline: Strategies to the rational design of multitarget ligands.

The role of histamine and acetylcholine in cognitive functions suggests that compounds able to increase both histaminergic and cholinergic neurotransmissions in the brain should be considered as promising therapeutic options. For this purpose, dual inhibitors of histamine H3 receptors (H3 R) and cholinesterases (ChEs) have been designed and assessed. In this context, this paper reviews the strategies used to obtain dual H3 R/ChEs ligands using multitarget design approaches. Hybrid compounds designed by linking tacrine or flavonoid motifs to H3 R antagonists were obtained with high affinity for both targets, and compounds designed by merging the H3 R antagonist pharmacophore with known anticholinesterase molecules were also reported. These reports strongly suggest that key modifications in the lipophilic region (including a second basic group) seem to be a strategy to reach novel compounds, allied with longer linker groups to a basic region. Some compounds have already demonstrated efficacy in memory models, although the pharmacokinetic and toxicity profile should be considered when designing further compounds. In conclusion, the key features to be considered when designing novel H3 R/ChEs inhibitors with improved pharmacological profile were herein summarized.

Molecular Determinants of the Kinetic Binding Properties of Antihistamines at the Histamine H1 Receptors.

The binding affinity of ligands for their receptors is determined by their kinetic and thermodynamic binding properties. Kinetic analyses of the rate constants of association and dissociation (kon and koff, respectively) of antihistamines have suggested that second-generation antihistamines have a long duration of action owing to the long residence time (1/koff) at the H1 receptors. In this study, we examined the relationship between the kinetic and thermodynamic binding properties of antihistamines, followed by an evaluation of the structural determinants responsible for their kinetic binding properties using quantitative structure-activity relationship (QSAR) analyses. We found that whereas the binding enthalpy and entropy might contribute to the increase and decrease, respectively, in the koff values, there was no significant relationship with the kon values. QSAR analyses indicated that kon and koff values could be determined by the descriptors FASA_H (water-accessible surface area of all hydrophobic atoms divided by total water-accessible surface area) and vsurf_CW2 (a 3D molecular field descriptor weighted by capacity factor 2, the ratio of the hydrophilic surface to the total molecular surface), respectively. These findings provide further insight into the mechanisms by which the kinetic binding properties of antihistamines are regulated by their thermodynamic binding forces and physicochemical properties.

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.

Roles of Lys191 and Lys179 in regulating thermodynamic binding forces of ligands to determine their binding affinity for human histamine H1 receptors.

Docking simulations based on the crystal structure of human histamine H1 receptors have predicted crucial roles of Lys1915.39 and Lys179ECL2, which exist at the entrance of the ligand-binding pocket, in increasing the H1-receptor selectivity for carboxylated second-generation antihistamines via electrostatic interaction. In this study, we evaluated the roles of Lys1915.39 and Lys179ECL2 in regulating the thermodynamic binding forces of non-carboxylated and carboxylated antihistamines that determine their binding affinity for human H1 receptors. The binding enthalpy and entropy of the 3 sets of non-carboxylated and corresponding carboxylated antihistamines (doxepin and olopatadine, desloratadine and loratadine, and terfenadine and fexofenadine, respectively) were estimated using the van't Hoff equation with the dissociation constants obtained from the displacement curves of the non-carboxylated and carboxylated antihistamines against the binding of [3H]mepyramine to the membrane preparations of Chinese hamster ovary cells expressing human H1 receptors at various temperatures, ranging from 4 °C to 37 °C. We found that the affinity for carboxylated antihistamines was lower than that for the corresponding non-carboxylated compounds due to lower enthalpy-dependent electrostatic binding forces and/or entropy-dependent hydrophobic binding forces. Mutations of Lys1915.39 and/or Lys179ECL2 to alanine mostly increased the binding affinity for antihistamines due to a variety of changes in both enthalpy- and entropy-dependent binding forces. These results suggest that Lys1915.39 and Lys179ECL2 may not contribute to selectively increasing the binding affinity for carboxylated antihistamines via electrostatic interaction, but that they can negatively modulate the binding affinity for non-carboxylated and carboxylated antihistamines non-selectively by affecting their electrostatic as well as hydrophobic binding forces.

The effects of antihistamine on the duration of the febrile seizure: A single center study with a systematic review and meta-analysis.

BACKGROUND: Several studies have investigated the potential effects of antihistamines on febrile seizure. However, these findings are inconsistent across the studies. METHOD: A retrospective observational study was conducted on a total of 434 consecutive patients aged between 6 months and 5 years with the diagnosis of febrile seizure. Patients with chronic medical conditions were excluded. Multivariable generalized linear models were conducted to ascertain the effects of antihistamine use on duration of febrile seizure. Also, we conducted a systematic review and meta-analyses of the medical literatures to calculate the pooled estimates using random effects models. RESULTS: The adjusted mean duration of febrile seizure in the antihistamine group was 4.9 min shorter than that in the non-user group (95% confidence interval (CI), 0.4-9.5). The risk of duration in febrile seizure >5 min among antihistamine users was also 0.83 times that among the non-users (95%CI, 0.58-1.19), whereas the risk of duration in febrile seizure >10 min among first-generation antihistamine users was 1.21 times that among non-users (95%CI, 0.69-2.13). According to the systematic review of the literature, 8 observational studies were included in the meta-analyses. Comparing to non-users, the antihistamine users had prolonged duration of febrile seizure by 1.07 min (95%CI, -1.13 to 3.27), elevated risk of duration in febrile seizure >5 min (Risk ratio, 1.16; 95%CI, 0.90-1.49), and similar duration from fever to febrile seizure onset (pooled mean difference, -0.01 h; -1.43 to 1.41), but these estimates were imprecise. Similar results were obtained when we stratified the data by types of antihistamine (first vs. second generation). CONCLUSIONS: Our study may indicate the effects of antihistamine on prolonging febrile seizure duration, but they are still controversial given the limited evidence, highly heterogeneous results, and concerns of the internal and external validities.

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists. Part 2. Versatile steroidal carboxamide derivatives.

To further proceed with our previous work, novel steroid-based histamine H3 receptor antagonists were identified and characterized. Using an 'amine-to-amide' modification strategy at position 17, in vitro and in vivo potent monoamino steroid derivatives were found during the lead optimization. Usage of the non-basic amide moiety resulted in beneficial effects both in activity and selectivity. The 15α-carboxamido derivative 10 was not only highly active at human and rat H3 receptors, but also showed negligible activity at rat muscarinic receptors. Furthermore, it proved to be considerably stable in human and rat microsomes and showed significant in vivo potency in the pharmacodynamic rat dipsogenia test and in the water-labyrinth cognitive model. Based on all of these considerations, compound 10 was appointed to be a preclinical candidate.

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 H1 R to H4 R. The H3 R 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 H3 R over the H4 R. Here, we describe their pharmacological properties at the human H1 R and H2 R in parallel with the H3 R, thus providing a full analysis of these compounds as histamine receptor ligands through reporter gene assays. Eight of the nine LINS01 compounds inhibited H3 R-induced histamine responses, but no inhibition of H2 R-induced responses was seen. Three compounds were weakly able to inhibit H1 R-induced responses. No agonist responses were seen to any of the compounds at any receptor. SAR analysis shows that the N-methyl group improves H3 R affinity while the N-phenyl group is detrimental. The methoxy derivative, LINS01009, had the highest affinity.

Binding of histamine to the H1 receptor-a molecular dynamics study.

Binding of histamine to the G-protein coupled histamine H1 receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp1073.32, Tyr1083.33, Thr1945.43, Asn1985.46, Trp4286.48, Tyr4316.51, Phe4326.52, and Phe4356.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 µs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp1073.32 for histamine binding.

Design and synthesis of histamine H3/H4 receptor ligands with a cyclopropane scaffold.

We previously designed and synthesized a series of histamine analogues with an imidazolylcyclopropane scaffold and identified potent non-selective antagonists for histamine H3 and H4 receptor subtypes. In this study, to develop H4 selective ligands, we newly designed and synthesized cyclopropane-based derivatives having an indole, benzimidazole, or piperazine structure, which are components of representative H4 selective antagonists such as JNJ7777120 and JNJ10191584. Among the synthesized derivatives, imidazolylcyclopropanes 12 and 13 conjugated with a benzimidazole showed binding affinity to the H3 and H4 receptors comparable to that of a well-known non-selective H3/H4 antagonist, thioperamide. These results suggest that the binding modes of the cyclopropane-based H3/H4 ligands in the H4 receptor can be different from those of the indole/benzimidazole-piperazine derivatives.

The antihistamine diphenhydramine is demethylated by anaerobic wastewater microorganisms.

While emerging pharmaceutical contaminants are monitored in wastewater treatment and the environment, there is little information concerning their microbial metabolites. The transformation of diphenhydramine by microorganisms in anaerobic digester sludge was investigated using anaerobic cultures amended with 1 mM diphenhydramine as the sole carbon source. Complete transformation of the parent compound to a persistent metabolite occurred within 191 days. Using GC/MS analysis, the metabolite was identified as N-desmethyl diphenhydramine. Loss of the parent compound diphenhydramine followed a first order rate constant of 0.013 day-1. There was no observed decrease in metabolite concentration even after a further 12 months of incubation, suggesting that the metabolite resists further degradation during wastewater treatment. Bacterial community diversity in the diphenhydramine transforming assay cultures showed enrichment in Comamonadaceae, Symbiobacteriaceae, Anaerolineaceae, and Prevotellaceae relative to unamended background controls. An anaerobic toxicity assay demonstrated that diphenhydramine has an inhibitory effect on both fermentative bacteria and methanogenic archaea in the wastewater community. In contrast, the metabolite N-desmethyl diphenhydramine partially suppressed methanogens but did not impact the fermenting community. To our knowledge, this is the first report of diphenhydramine metabolism by a bacterial community. The limited transformation of diphenhydramine by wastewater microorganisms indicates that N-desmethyl diphenhydramine will enter the environment along with unmetabolized diphenhydramine.

Genotoxicity of 4-(piperazin-1-yl)-8-(trifluoromethyl)pyrido[2,3-e][1,2,4] triazolo[4,3-a]pyrazine, a Potent H4 Receptor Antagonist for the Treatment of Allergy: Evidence of Glyoxal Intermediate Involvement.

BACKGROUND: 4-(piperazin-1-yl)-8-(trifluoromethyl)pyrido[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (1) is a small-molecule which demonstrated a sub-nM inhibitory potency toward the histamine H4 receptor (H4R). However, it was found to be mutagenic in an in vitro Ames assay. Metabolic bioactivation of 1 could potentially arise from the piperazine moiety by forming reactive intermediates such as glyoxal, aldehyde-imine and/or iminium ion, which could all lead to genotoxicity. The aim of this study was to investigate bioactivation of 1 to determine the potential causes of the genotoxicity and mitigate liabilities in this scaffold. METHODS: 1 was investigated for its genotoxicity in phenobarbital and ß-naphthoflavone induced Sprague Dawley rat liver S9 fractions. Trapping agents such as o-phenylenediamine was used postincubation. RESULTS: Following metabolic profiling of 1, two oxidative metabolites were observed and identified in phenobarbital- and ß -naphthoflavone induced Sprague Dawley rat liver S9 fractions. Metabolic pathway of 1 was primarily mediated by the metabolism of the piperazine moiety. The trapped glyoxal was identified by using high resolution LC-MS instrument. Structural characterization of the trapped glyoxal was determined by comparison of retention time, accurate mass measurement and Collision Induced Dissociation (CID) spectra to authentic standard. CONCLUSION: In the present investigation, a novel method was developed to trap glyoxal, which may potentially be liberated from piperazine moiety. These findings led to modifications on the piperazine ring to mitigate the bioactivation pathways leading to mutagenicity. Subsequently, the next generation compounds with modified piperazine moiety, retained H4R inhibitory potency in vitro and were not genotoxic in the Ames mutagenicity assay.

Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method.

Virtual screening offers an efficient alternative to high-throughput screening in the identification of pharmacological tools and lead compounds. Virtual screening is typically based on the matching of target structures or ligand pharmacophores to commercial or in-house compound catalogues. This study provides the first proof-of-concept for our recently reported method where pharmacophores are instead constructed based on the inference of residue-ligand fragments from crystal structures. We demonstrate its unique utility for G protein-coupled receptors, which represent the largest families of human membrane proteins and drug targets. We identified five neutral antagonists and one inverse agonist for the histamine H3 receptor with potencies of 0.7-8.5 µM in a recombinant receptor cell-based inositol phosphate accumulation assay and validated their activity using a radioligand competition binding assay. H3 receptor antagonism is of large therapeutic value and our ligands could serve as starting points for further lead optimisation. The six ligands exhibit four chemical scaffolds, whereof three have high novelty in comparison to the known H3 receptor ligands in the ChEMBL database. The complete pharmacophore fragment library is freely available through the GPCR database, GPCRdb, allowing the successful application herein to be repeated for most of the 285 class A GPCR targets. The method could also easily be adapted to other protein families.