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).

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.

An in-silico evaluation of COVID-19 main protease with clinically approved drugs.

A novel strain of coronavirus, namely, SARS-CoV-2 identified in Wuhan city of China in December 2019, continues to spread at a rapid rate worldwide. There are no specific therapies available and investigations regarding the treatment of this disease are still lacking. In order to identify a novel potent inhibitor, we performed blind docking studies on the main virus protease Mpro with eight approved drugs belonging to four pharmacological classes such as: anti-malarial, anti-bacterial, anti-infective and anti-histamine. Among the eight studied compounds, Lymecycline and Mizolastine appear as potential inhibitors of this protease. When docked against Mpro crystal structure, these two compounds revealed a minimum binding energy of -8.87 and -8.71 kcal/mol with 168 and 256 binding modes detected in the binding substrate pocket, respectively. Further, to study the interaction mechanism and conformational dynamics of protein-ligand complexes, Molecular dynamic simulation and MM/PBSA binding free calculations were performed. Our results showed that both Lymecycline and Mizolastine bind in the active site. And exhibited good binding affinities towards target protein. Moreover, the ADMET analysis also indicated drug-likeness properties. Thus it is suggested that the identified compounds can inhibit Chymotrypsin-like protease (3CLpro) of SARS-CoV-2.

Assessment of Pharmacologic Ingredients in Common Over-the-Counter Sinonasal Medications.

Importance: Sinonasal remedies are the most frequently purchased category of over-the-counter (OTC) drugs in the United States. A variety of options for relief are available under proprietary names, although the actual number of available options may not be readily appreciated by the consumer or the clinician. Objective: To determine the prevalence of specific ingredients in OTC sinonasal products. Design, Setting, and Participants: This cross-sectional study took physical inventory of brand-name and generic OTC drugs marketed as sinus, cold, allergy, or nasal remedies. Retail pharmacies in New Orleans, Louisiana, commercial websites, and the Drugs, Herbs and Supplements section of MedlinePlus and drugs.com were searched. Data were collected and analyzed from July 1 to 31, 2018. Main Outcomes and Measures: Frequency of active ingredients in OTC formulations. Results: Five pharmacies were visited to identify 18 brands, for which the commercial websites were then searched. The 14 most common brands represented 211 unique products. Only 8 unique nonanalgesic ingredients were identified among these products, with many products sold under the same brand name and with the same active ingredient. Phenylephrine hydrochloride, dextromethorphan hydrobromide, pseudoephedrine hydrochloride, guaifenesin, chlorpheniramine maleate, brompheniramine maleate, diphenhydramine hydrochloride, and doxylamine succinate were the common active ingredients, with all available OTC sinonasal remedies consisting of 1 or more of these ingredients. The frequency of occurrence of each ingredient ranged from 10 to 261 different products. Combinations of 2, 3, or 4 active ingredients occurred frequently in OTC sinonasal products. Conclusions and Relevance: These findings suggest that proliferation of brand extension products under a common name is pervasive. Clinicians should be aware of the large array of redundant OTC formulations and lack of specificity when discussing brand-name sinonasal remedies with their patients.

Asymmetric Catalysis upon Helically Chiral Loratadine Analogues Unveils Enantiomer-Dependent Antihistamine Activity.

Analogues of the conformationally dynamic Claritin (loratadine) and Clarinex (desloratadine) scaffolds have been enantio- and chemoselectively N-oxidized using an aspartic acid containing peptide catalyst to afford stable, helically chiral products in up to >99:1 er. The conformational dynamics and enantiomeric stability of the N-oxide products have been investigated experimentally and computationally with the aid of crystallographic data. Furthermore, biological assays show that rigidifying the core structure of loratadine and related analogues through N-oxidation affects antihistamine activity in an enantiomer-dependent fashion. Computational docking studies illustrate the observed activity differences.

Covalent Inhibition of the Histamine H3 Receptor.

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H3 receptor (H3R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H3R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H3R was validated with washout experiments and leads to inverse agonism on H3R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H3R conformation and to study the consequences of prolonged inhibition of the H3R.

Dual lysosomal-mitochondrial targeting by antihistamines to eradicate leukaemic cells.

BACKGROUND: Despite great efforts to identify druggable molecular targets for AML, there remains an unmet need for more effective therapies. METHODS: An in silico screening was performed using Connectivity Maps to identify FDA-approved drugs that may revert an early leukaemic transformation gene signature. Hit compounds were validated in AML cell lines. Cytotoxic effects were assessed both in primary AML patient samples and healthy donor blood cells. Xenotransplantation assays were undertaken to determine the effect on engraftment of hit compounds. The mechanism of action responsible for the antileukaemic effect was studied focussing on lysosomes and mitochondria. FINDINGS: We identified a group of antihistamines (termed ANHAs) with distinct physicochemical properties associated with their cationic-amphiphilic nature, that selectively killed leukaemic cells. ANHAs behaved as antileukaemic agents against primary AML samples ex vivo, sparing healthy cells. Moreover, ANHAs severely impaired the in vivo leukaemia regeneration capacity. ANHAs' cytotoxicity relied on simultaneous mitochondrial and lysosomal disruption and induction of autophagy and apoptosis. The pharmacological effect was exerted based on their physicochemical properties that permitted the passive targeting of both organelles, without the involvement of active molecular recognition. INTERPRETATION: Dual targeting of lysosomes and mitochondria constitutes a new promising therapeutic approach for leukaemia treatment, supporting the further clinical development. FUND: This work was funded by the Fundación Mutua Madrileña (RMR), CaixaImpulse (RMR), the Spanish Ministry of Economy (RMR), the Josep Carreras International Leukaemia Foundation (RMR), l'Obra Social "La Caixa" (RMR), and Generalitat de Catalunya (IJC).

Cell Death Induced by Cationic Amphiphilic Drugs Depends on Lysosomal Ca2+ Release and Cyclic AMP.

Repurposing cationic amphiphilic drugs (CAD) for cancer treatment is emerging as an attractive means to enhance the efficacy of chemotherapy. Many commonly used CADs, including several cation amphiphilic antihistamines and antidepressants, induce cancer-specific, lysosome-dependent cell death and sensitize cancer cells to chemotherapy. CAD-induced inhibition of lysosomal acid sphingomyelinase is necessary, but not sufficient, for the subsequent lysosomal membrane permeabilization and cell death, while other pathways regulating this cell death pathway are largely unknown. Prompted by significant changes in the expression of genes involved in Ca2+ and cyclic AMP (cAMP) signaling pathways in CAD-resistant MCF7 breast cancer cells, we identified here an early lysosomal Ca2+ release through P2X purinergic receptor 4 (P2RX4) and subsequent Ca2+- and adenylyl cyclase 1 (ADCY1)-dependent synthesis of cAMP as a signaling route mediating CAD-induced lysosomal membrane permeabilization and cell death. Importantly, pharmacologic and genetic means to increase cellular cAMP levels either by activating cAMP-inducing G-protein-coupled receptors (GPR3 or ß2 adrenergic receptor) or ADCY1, or by inhibiting cAMP-reducing guanine nucleotide-binding protein G(i) subunit α2, C-X-C motif chemokine receptor type 4, or cAMP phosphodiesterases, sensitized cancer cells to CADs. These data reveal a previously unrecognized lysosomal P2RX4- and ADCY1-dependent signaling cascade as a pathway essential for CAD-induced lysosome-dependent cell death and encourage further investigations to find the most potent combinations of CADs and cAMP-inducing drugs for cancer therapy.

Loratadine self-microemulsifying drug delivery systems (SMEDDS) in combination with sulforaphane for the synergistic chemoprevention of pancreatic cancer.

Pancreatic cancer (PC), currently the third leading cause of cancer-related deaths in the USA, is projected to become the second leading cause, behind lung cancer, by 2020. The increasing incidence, low survival rate, and limited treatment opportunities necessitate the use of alternative approaches such as chemoprevention, to tackle PC. In this study, we report significant synergistic chemoprevention efficacy for the first time from a low-dose combination of a classical antihistaminic drug, Loratadine (LOR) and a neutraceutical compound, Sulforaphane (SFN) using a self-microemulsifying drug delivery system (SMEDDS) formulation. The formulation was developed using Quality by Design approach (globule size, 95.13 ± 7.9 nm; PDI, 0.17 ± 0.04) and revealed significant (p < 0.05) enhancement in the in vitro dissolution profile confirming the enhanced solubility of BCS class II drug LOR with SMEDDS formulation. The LOR-SFN combination revealed ~ 40-fold reduction in IC50 concentration compared to LOR alone in MIA PaCa-2 and Panc-1 cell lines respectively, confirming the synergistic enhancement in chemoprevention. Further, the nanoformulation resulted in ~ 7-fold and ~ 11-fold reduction in IC50 values compared to LOR-SFN combination. Hence, our studies successfully demonstrate that a unique low-dose combination of LOR encapsulated within SMEDDs with SFN shows significantly enhanced chemopreventive efficacy of PC.

Phytoconstituents from Polyscias guilfoylei leaves with histamine-release inhibition activity.

Phytochemical investigation of Polyscias guilfoylei leaves extract (PGE) led to the isolation of nine compounds, that is, ent-labda-8(17),13-diene-15,18-diol (1), stigmasterol (2), spinasterol (3), N-(1,3-dihydroxyoctadecan-2-yl) palmitamide (4), panaxydiol (5), 3-O-ß-d-glucopyranosylstigmasta-5,22-diene-3-ß-ol (6), (8Z)-2-(2 hydroxypentacosanoylamino) octadeca-8-ene-1,3,4-triol (7), 4-hydroxybenzoic acid (8), and tamarixetin 3,7-di-O-α-L-rhamnopyranoside (9). Compound 4 is reported in this study for the first time in nature whereas compound 9 is reported for the second time. Structural elucidation of the compounds was carried out using Nuclear Magnetic Resonance and Electrospray Ionization coupled with Mass Spectrometry spectroscopic analyses. PGE and compounds 4 and 9 exhibited weak cytotoxicity against both MCF-7 and HCT-116 cell lines using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay. The antimicrobial activity of PGE and compounds 4 and 9 was evaluated using the agar diffusion method. Escherichia coli was the most susceptible Gram-negative bacteria toward PGE with a minimum inhibitory concentration value of 9.76 µg/mL, whereas compounds 4 and 9 did not show any antimicrobial activity. Compound 4 exhibited promising inhibition of histamine release using U937 human monocytes with an IC50 value of 38.65 µg/mL.

Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase.

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z' score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure-activity relationships.

Conversion of Curcumin into Heterocyclic Compounds as Potent Anti-diabetic and Anti-histamine Agents.

Potential biologically active derivatives of the curcumin were prepared by the cyclocondensation reaction cyclohexanone 2, imino pyrimidine 3, pyrmidinones 4, thiopyrimidine 6 and pyranone 5, 7 when treated with acetylacetone, guanidine, ureaethylcyanoacetate, thiourea and ethylacetoacetate, respectively. The structures of compounds (2-7) were elucidated by means of microanalysis as well as spectral measurements such as IR, 1H-NMR, MS. The anti-diabetic potential of curcumin derivatives were evaluated by assessing amylase inhibition assay, also inhibition of histamine release activity of curcumin derivatives were assessed by U937 human monocytes. The results for amylase inhibition activity revels that the curcumin inhibits α-amylase in a concentration dependent manner. Compounds 4 and 5 exhibited significant inhibitory activity against amylase enzyme and was comparable with that of acrabose. Also, compounds 5, 6 and 7 exhibited significant inhibitory activity against histamine. Our results concluded that curcumin pyrmidinones and pyranone derivatives have highly effects as anti-diabetic and anti-histamine activities.

A fully validated method for the simultaneous determination of 11 antihistamines in breast milk by gas chromatography-mass spectrometry.

Antihistamines are excreted into breast milk in small amounts; however, there are no adequate published studies or data concerning their effects on newborns and safety during breastfeeding. Thus, the development of sensitive and specific methodologies for the determination of antihistamines in breast milk is critical. A simple and sensitive GC-MS method for the simultaneous determination of 11 antihistamines (diphenhydramine, orphenadrine, chlorpheniramine, dimethindene, meclozine, hydroxyzine, loratadine, desloratadine, cetirizine, rupatadine and ebastine) in breast milk was developed and validated. The antihistamines were solid-phase extracted and derivatized with acetic anhydride and n-propanol. Diazepam-d5 , hydroxyzine-d4 and cetirizine-d8 were used as internal standards. Absolute recovery values for all analytes ranged from 70.5 to 120.0%, while the limits of detection and quantification for all analytes were 1.50 and 5.00 ng/mL, respectively. All calibration curves were linear (R2 ≥ 0.990) within the range 5.00-1000.0 ng/mL. Accuracy (Er ) ranged between -7.6 and 7.0%, while precision (RSD) was <12% for all antihistamines. The developed method is suitable for the investigation of antihistamine-related clinical cases, as well as for pharmacokinetic and breastfeeding safety studies.

An overview of quinazolines: Pharmacological significance and recent developments.

Most of the drugs and pharmacologically relevant molecules possess heterocyclic ring structures and presence of hetero atoms or groupings divulges privileged specificities in their pharmacological targets. Especially the heterocyclic systems, quinazoline is a biologically imperative scaffold known to be linked with several pharmacological activities. Some of the protuberant pharmacological responses attributed to this system are analgesic, anti-inflammatory, anti-convulsant, sedative-hypnotic, anti-histaminic, anti-hypertensive, anti-cancer, anti-microbial, anti-tubercular and anti-viral activities. This multiplicity in the pharmacological response contours of quinazoline has attracted the consideration of medicinal chemists to explore this system to its multiple potential against numerous activities. Several of these synthetic and pharmacological investigations have been successively studied for structure-activity relationship (SAR) to correlate the particular structural features for their pharmacological target. The emerging understanding of quinazoline derivatives on their pharmacological target offer opportunities for novel therapeutics. This review principally emphases on the medicinal chemistry aspects including drug design, structure-activity relationships (SARs), and mechanism of actions of quinazoline derivatives. This review gives detailed attention on in vitro and in vivo pharmacological activities of quinazoline and its analogs in the perspective of drug discovery and its development.

Histamine and Histaminergic Receptors in Colorectal Cancer: From Basic Science to Evidence-based Medicine.

BACKGROUND: Histamine is an imidazolic compound performing a crucial function in the pathogenesis of inflammation. Several studies have also emphasized its pro-carcinogenic effect in colorectal cancer (CRC). OBJECT: In fact, increased histamine levels have been observed in CRC and a decreased catabolism of this molecule is typical of colorectal adenomas. Additional data have demonstrated that CRC is characterized by an altered balance of histamine receptors (HRs); in fact, HR1 and HR4 are down-regulated in CRC, while HR2 is overexpressed. METHOD: Based on this evidence, we reviewed several studies investigating the role of HR2 antagonists (HR2A), such as cimetidine in CRC. RESULTS: From a clinical point of view, HR2A may prolong the survival rates of patients with CRC, and a recent meta-analysis seems to confirm this finding. From a biological perspective, it has been demonstrated that HR2A could have a beneficial effect on CRC for many reasons: i) promotion of peri-tumoral lymphocyte growth and improvement of immune response against the tumor, ii) suppression of adhesion molecules which might favor metastasis, iii) anti-angiogenetic activity (reduction of VEGF), iv) increased production of some cytokines which may counteract tumor growth, such as tumor necrosis factor (TNF) alpha, interleukin (IL)-10 and IL-15. On the contrary, HR1 antagonists did not demonstrate any beneficial effect on CRC. Therefore, it is presumable that histamine could be a relevant player in the development of CRC, but its effect might be mediated by an imperfect homeostasis of its receptors. CONCLUSION: In this scenario, HR2A could inhibit carcinogenesis whereas HR2 might act as a pro-carcinogenetic, while HR1 and HR4, being suppressed in CRC, may antagonize neoplastic development.

Identification of a coumarin-based antihistamine-like small molecule as an anti-filoviral entry inhibitor.

Filoviruses, consisting of Ebola virus, Marburg virus and Cuevavirus, cause severe hemorrhagic fevers in humans with high mortality rates up to 90%. Currently, there is no approved vaccine or therapy available for the prevention and treatment of filovirus infection in humans. The recent 2013-2015 West African Ebola epidemic underscores the urgency to develop antiviral therapeutics against these infectious diseases. Our previous study showed that GPCR antagonists, particularly histamine receptor antagonists (antihistamines) inhibit Ebola and Marburg virus entry. In this study, we screened a library of 1220 small molecules with predicted antihistamine activity, identified multiple compounds with potent inhibitory activity against entry of both Ebola and Marburg viruses in human cancer cell lines, and confirmed their anti-Ebola activity in human primary cells. These small molecules target a late-stage of Ebola virus entry. Further structure-activity relationship studies around one compound (cp19) reveal the importance of the coumarin fused ring structure, especially the hydrophobic substituents at positions 3 and/or 4, for its antiviral activity, and this identified scaffold represents a favorable starting point for the rapid development of anti-filovirus therapeutic agents.

Development of a buccal doxepin platform for pain in oral mucositis derived from head and neck cancer treatment.

This study describes the development of semisolid formulations containing doxepin (DOX) for pain relief in oral mucositis, frequently related to chemotherapy and/or radiotherapy treatments in patients with head and neck cancer. Chemical permeation enhancers were evaluated and selected according to the results obtained from rheological studies, drug release, and drug permeation and retention through buccal mucosa. Finally, the selected formulation was compared in vivo, with a reference DOX mouthwash, whose clinical efficacy had been previously reported. The obtained findings showed that an orabase® platform loading transcutol® (10%) and menthol (5%) for the buccal vehiculization of DOX exhibited a decreased elastic and viscous behavior improving its application. The main drug release mechanism could be considered as diffusion according to Higuchi model. Obtained DOX permeation rates were considered optimal for an analgesic effect and far below to an antidepressant activity. Similar in vivo plasma concentrations were found for the semisolid formulation and the reference mouthwash. However, DOX amounts retained in the mucosa of animals for the semisolid formulation were higher than the reference, which let us hypostatize even stronger potential local therapeutic effect with additional advantages such as, mucoadhesive properties, absence of alcohol, some degree of freshness, as well as, drug palatability improvement.

Effect of H4R antagonist N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamides and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole on histamine and 4-methylhistamine-induced mast cell response.

CONTEXT: The histamine plays a decisive role in acute and chronic inflammatory responses and is regulated through its four types of distinct receptors designated from H1 to H4. Recently histamine 4 receptor (H4R) antagonists have been reported to possess various pharmacological effects against various allergic diseases. OBJECTIVE: To investigate the inhibitory effect of N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamide (Compound A) and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole (Compound L) on H4R-mediated calcium mobilization, cytokine IL-13 production, ERK1/2, Akt and NF-κB activation in human mastocytoma cells-1 (HMC-1). MATERIALS AND METHODS: Compounds A and L were synthesized chemically and their inhibitory effect on intracellular calcium release was analyzed by Fluo-4 calcium assay, cytokine measurement through ELISA and activation of signaling molecules by western blot. RESULTS: Pre-treatment with compounds A and L significantly reduced the H4R-mediated intracellular calcium release. Histamine and 4-methylhistamine (4-MH) induced Th2 cytokine IL-13 production in HMC-1 cells, was inhibited by compound A (77.61%, 74.25% at 1 µM concentration) and compound L (79.63%, 81.70% at 1 µM concentration). Furthermore, histamine induced the phosphorylation of ERK1/2, Akt and NF-κB was suppressed by compounds A and L at varying levels, ERK1/2 (88%, 86%), Akt (88%, 89%) and NF-κB (89%, 87%) in HMC-1 cells. DISCUSSION AND CONCLUSIONS: Taken together these data demonstrate that compound A and compound L may block H4R-mediated downstream signaling events.

Probing an artificial polypeptide receptor library using a series of novel histamine H3 receptor ligands.

An artificial polypeptide receptor (APR) library was created by using the self-organization of N-lipidated peptides attached to cellulose via m-aminophenylamino-1,3,5-triazine. The response of the library was probed using a series of novel H3 receptor ligands. Since no guidelines on how to design an APRs selective vs certain receptor types exist, a diverse set of amino acids (Ala, Trp, Pro, Glu, His, Lys and Ser) were used and coupled with one of three gating fatty acids (palmitic, ricinoleic or capric). A competitive adsorption-desorption of an appropriate reporter dye was used for the indirect visualization of the interactions of guests with particular receptors. The resulted library response to individual inhibitors was then arranged in a matrix, preprocessed and analyzed using the principal component analysis (PCA) and partial least squares (PLS) method. The most important conclusion obtained from the PCA analysis is that the library differentiates the probed compounds according to the lipophilicity of the gating unit. The PC3 with a dominant absolute contribution of the receptors containing Glu allowed for the best separation of the ligands with respect to their activity. This conclusion is in agreement with the fact that Glu 206 is a genuine ligand counterpart in the natural histamine receptor.