[3H]UR‐DE257: Development of a Tritium‐Labeled Squaramide‐Type Selective Histamine H2 Receptor Antagonist

A series of new piperidinomethylphenoxypropylamine‐type histamine H₂ receptor (H₂R) antagonists with different substituted “urea equivalents” was synthesized and characterized in functional in vitro assays. Based on these data as selection criteria, radiosynthesis of N‐[6‐(3,4‐dioxo‐2‐{3‐[3‐(piperidin‐1‐ylmethyl)phenoxy]propylamino}cyclobut‐1‐enylamino)hexyl]‐(2,3‐³H₂)propionic amide ([³H]UR‐DE257) was performed. The radioligand (specific activity: 63 Ci mmol^?1) had high affinity for human, rat, and guinea pig H₂R (hH₂R, Sf9 cells: K_d, saturation binding: 31 nM , kinetic studies: 20 nM ). UR‐DE257 revealed high H₂R selectivity on membranes of Sf9 cells, expressing the respective hH_xR subtype (K_i values: hH₁R: >10 000 nM , hH₂R: 28 nM , hH₃R: 3800 nM , hH₄R: >10 000 nM ). In spite of insurmountable antagonism, probably due to rebinding of [³H]UR‐DE257 to the H₂R (extended residence time), the title compound proved to be a valuable pharmacological tool for the determination of H₂R affinities in competition binding assays.     

Synthesis and Characterization of New Bivalent Agents as Melatonin- and Histamine H3-Ligands

Melatonin is an endogenous molecule involved in many pathophysiological processes. In addition to the control of circadian rhythms, its antioxidant and neuroprotective properties have been widely described. Thus far, different bivalent compounds composed by a melatonin molecule linked to another neuroprotective agent were synthesized and tested for their ability to block neurodegenerative processes in vitro and in vivo. To identify a novel class of potential neuroprotective compounds, we prepared a series of bivalent ligands, in which a prototypic melatonergic ligand is connected to an imidazole-based H₃ receptor antagonist through a flexible linker. Four imidazolyl-alkyloxy-anilinoethylamide derivatives, characterized by linkers of different length, were synthesized and their binding affinity for human MT₁, MT₂ and H₃ receptor subtypes was evaluated. Among the tested compounds, 14c and 14d, bearing a pentyl and a hexyl linker, respectively, were able to bind to all receptor subtypes at micromolar concentrations and represent the first bivalent melatonergic/histaminergic ligands reported so far. These preliminary results, based on binding affinity evaluation, pave the way for the future development of new dual-acting compounds targeting both melatonin and histamine receptors, which could represent promising therapeutic agents for the treatment of neurodegenerative pathologies.     

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation,Signaling and Interactions with GRKs and β-Arrestins

The histamine H₁ receptor (H₁R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H₁R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H₁R signals via heterotrimeric G_q proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H₁R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H₁R signaling through heterotrimeric G_q proteins, second messengers (inositol 1,4,5-triphosphate and Ca²⁺), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H₁R over G_q biosensor activation.     

Synthesis,Pharmacological Evaluation,and Docking Studies of Novel Pyridazinone‐Based Cannabinoid Receptor Type 2 Ligands

In recent years, cannabinoid type 2 receptors (CB₂R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB₂R are devoid of the psychoactive effects typically observed for CB₁R ligands. Based on our recent studies on a class of pyridazinone 4‐carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure–activity relationships for this promising scaffold with the aim to develop potent CB₂R ligands. In binding assays, two of the new synthesized compounds [6‐(3,4‐dichlorophenyl)‐2‐(4‐fluorobenzyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2,3‐dihydropyridazine‐4‐carboxamide ( 2 ) and 6‐(4‐chloro‐3‐methylphenyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2‐pentyl‐2,3‐dihydropyridazine‐4‐carboxamide ( 22 )] showed high CB₂R affinity, with K_i values of 2.1 and 1.6 nm , respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB₂R inverse agonists. Compound 22 displayed the highest CB₂R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle‐switch residue, W6.48.     

KSK-74: Dual Histamine H3 and Sigma-2 Receptor Ligand with Anti-Obesity Potential

Many studies involving compounds that enhance histamine release, such as histamine H₃ receptor (H₃R) antagonists, have shown efficacy in inhibiting weight gain, but none have passed clinical trials. As part of the search for H₃ receptor ligands that have additional properties, the aim of this study is to evaluate the activity in the reduction in weight gain in a rat model of excessive eating, as well as the impact on selected metabolic parameters, and the number and size of adipocytes of two new H₃R antagonists, KSK-60 and KSK-74, which also exert a significant affinity at the sigma-2 receptor. Compounds KSK-60 and KSK-74 are homologues and the elongation of the distal part of the molecule resulted in an approximate two-fold reduction in affinity at H₃R, but simultaneously an almost two-fold increase in affinity at the sigma-2 receptor. Animals fed palatable feed and receiving KSK-60 or KSK-74 both at 10 mg/kg b.w. gained significantly less weight than animals in the control obese group. Moreover, KSK-74 significantly compensated for metabolic disturbances that accompany obesity, such as an increase in plasma triglyceride, resistin, and leptin levels; improved glucose tolerance; and protected experimental animals against adipocyte hypertrophy. Furthermore, KSK-74 inhibited the development of inflammation in obesity-exposed adipose tissue. The in vivo pharmacological activity of the tested ligands appears to correlate with the affinity at the sigma-2 receptors; however, the explanation of this phenomenon requires further and extended research.     

Involvement of Histamine H3 Receptor Agonism in Premature Ejaculation Found by Studies in Rats

Several of the drugs currently available for the treatment of premature ejaculation (PE) (e.g., local anesthetics or antidepressants) are associated with numerous safety concerns and exhibit weak efficacy. To date, no therapeutics for PE have been approved in the United States, highlighting the need to develop novel agents with sufficient efficacy and fewer side effects. In this study, we focused on the histamine H₃ receptor (H₃R) as a potential target for the treatment of PE and evaluated the effects of imetit (an H₃R/H₄R agonist), ciproxifan (an H₃R antagonist), and JNJ-7777120 (an H₄R antagonist) in vivo. Our in vivo electrophysiological experiments revealed that imetit reduced mechanical stimuli-evoked neuronal firing in anesthetized rats. This effect was inhibited by ciproxifan but not by JNJ-7777120. Subsequently, we evaluated the effect of imetit using a copulatory behavior test to assess ejaculation latency (EL) in rats. Imetit prolonged EL, although this effect was inhibited by ciproxifan. These findings indicate that H₃R stimulation suppresses mechanical stimuli-evoked neuronal firing in the spinal–penile neurotransmission system, thereby resulting in prolonged EL. To our knowledge, this is the first report to describe the relationship between H₃R and PE. Thus, H₃R agonists may represent a novel treatment option for PE.     

Interaction of the σ2 Receptor Ligand PB28 with the Human Nucleosome: Computational and Experimental Probes of Interaction with the H2A/H2B Dimer

Sigma‐2 (σ₂) binding sites are an emerging target for anti‐neoplastic agents due to the strong apoptotic effect exhibited by σ₂ agonists in vitro and the overexpression of these sites in tumor cells. Nonetheless, no σ₂ receptor protein has been identified. Affinity chromatography using the high‐affinity σ₂ ligand PB28 and human SK‐N‐SH neuroblastoma cells was previously utilized to identify σ₂ ligand binding proteins, specifically histones H1, H2A, H2B, and H3.3a. To rationalize this finding, homology modeling and automated docking studies were employed to probe intermolecular interactions between PB28 and human nucleosomal proteins. These studies predicted interaction of PB28 with the H2A/H2B dimer at a series of sites previously found to be implicated in chromatin compaction and nucleosomal assembly. To experimentally verify this prediction, a competitive binding assay was performed on the reconstituted H2A/H2B dimer using [³H]PB28 as radioligand, and an IC₅₀ value of 0.50 nM was determined for PB28 binding. In addition, [³H]PB28 was found to accumulate with up to a fivefold excess in nuclear fractions over cytosolic fractions of SK‐N‐SH and MCF7 cells, indicating that PB28 is capable of entering the nucleus to interact with histone proteins. In conjunction with computational results, these data suggest that PB28 may exert its cytotoxic effect through direct interaction with nuclear material.     

Bivalent Argininamide‐Type Neuropeptide Y Y1 Antagonists Do Not Support the Hypothesis of Receptor Dimerisation

Bivalent ligands are potential tools to investigate the dimerisation of G‐protein‐coupled receptors. Based on the (R)‐argininamide BIBP 3226, a potent and selective neuropeptide Y Y₁ receptor (Y₁R) antagonist, we prepared a series of bivalent Y₁R ligands with a wide range of linker lengths (8–36 atoms). Exploiting the high eudismic ratio (>1000) of the parent compound, we synthesised sets of R,R‐, R,S‐ and S,S‐configured bivalent ligands to gain insight into the “bridging” of two Y₁Rs by simultaneous interaction with both binding sites of a putative receptor dimer. Except for the S,S isomers, the bivalent ligands are high‐affinity Y₁R antagonists, as determined by Ca²⁺ assays on HEL cells and radioligand competition assays on human Y₁R‐expressing SK‐N‐MC and MCF‐7 cells. Whereas the R,R enantiomers are most potent, no marked differences were observed relative to the corresponding meso forms. The difference between R,R and R,S diastereomers was most pronounced (about sixfold) in the case of the Y₁R antagonist containing a spacer of 20 atoms in length. Among the R,R enantiomers, linker length and structural diversity had little effect on Y₁R affinity. Although the bivalent ligands preferentially bind to the Y₁R, the selectivity toward human Y₂, Y₄, and Y₅ receptors was markedly lower than that of the monovalent argininamides. The results of this study neither support the presence of Y₁R dimers nor the simultaneous occupation of both binding pockets by the twin compounds. However, as the interaction with Y₁R dimers cannot be unequivocally ruled out, the preparation of a bivalent radioligand is suggested to determine the ligand–receptor stoichiometry. Aiming at such radiolabelled pharmacological tools, prototype twin compounds were synthesised, containing an N‐propionylated amino‐functionalised branched linker (K_i≥18 nM ), a tritiated form of which can be easily prepared.     

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D_2S and D₃ belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D_2S and D₃ receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate K_D-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.     

Insight into the Interactions between Novel Coumarin Derivatives and Human A3 Adenosine Receptors

A study focused on the discovery of new chemical entities based on the 3‐arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A₁, A_2A, and A₃) and adenylyl cyclase activity (A_2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A₃AR ligands, with 3‐(4′‐methylphenyl)‐8‐(2‐oxopropoxy)coumarin ( 12 ) being the most potent (K_i=634 nM ). None of the compounds showed affinity for the A_2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3‐arylcoumarin scaffold composes a novel and promising class of A₃AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.     

MS Binding Assays for D1 and D5 Dopamine Receptors

MS Binding Assays are a label‐free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D₁ and D₅ dopamine receptors. A highly sensitive, rapid and robust LC–ESI‐MS/MS quantification method for the selective D₁ dopamine receptor antagonist SCH23390 ((5R)‐8‐chloro‐3‐methyl‐5‐phenyl‐1,2,4,5‐tetrahydro‐3‐benzazepin‐7‐ol) was established and validated, using its 8‐bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D₁ and D₅ receptors. Following the concept of MS Binding Assays, saturation experiments for D₁ and D₅ receptors were performed, as well as competition experiments for D₁ receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D₁ and D₅ receptors are well‐suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.     

Indoloquinolizidine–Peptide Hybrids as Multiple Agonists for D1 and D2 Dopamine Receptors

Multiple‐specificity ligands are considered promising pharmacological tools that may show higher efficacy in the treatment of diseases for which the modulation of a single target is therapeutically inadequate. We prepared a set of novel ligands for D₁ and D₂ dopamine receptors by combining two indolo[2,3‐a]quinolizidine scaffolds with various tripeptide moieties. The binding and functional properties of these molecules were determined by radioligand binding studies in brain striatum membranes and by intracellular cAMP production assays in cells expressing different dopamine receptor subtypes. Some indoloquinolizidine–peptide hybrids, mainly with the trans configuration, showed dual agonist activity at both D₁ and D₂ dopamine receptors and may therefore be useful for testing the therapeutic potential of multivalent drugs on these targets.     

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, E_max) 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.     

Design,Radiosynthesis and Preliminary Biological Evaluation in Mice of a Brain-Penetrant 18F-Labelled σ2 Receptor Ligand

The σ₂ receptor (transmembrane protein 97), which is involved in cholesterol homeostasis, is of high relevance for neoplastic processes. The upregulated expression of σ₂ receptors in cancer cells and tissue in combination with the antiproliferative potency of σ₂ receptor ligands motivates the research in the field of σ₂ receptors for the diagnosis and therapy of different types of cancer. Starting from the well described 2-(4-(1H-indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline class of compounds, we synthesized a novel series of fluorinated derivatives bearing the F-atom at the aromatic indole/azaindole subunit. RM273 (2-[4-(6-fluoro-1H-pyrrolo[2,3-b]pyridin-1-yl)butyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) was selected for labelling with ¹⁸F and evaluation regarding detection of σ₂ receptors in the brain by positron emission tomography. Initial metabolism and biodistribution studies of [¹⁸F]RM273 in healthy mice revealed promising penetration of the radioligand into the brain. Preliminary in vitro autoradiography on brain cryosections of an orthotopic rat glioblastoma model proved the potential of the radioligand to detect the upregulation of σ₂ receptors in glioblastoma cells compared to healthy brain tissue. The results indicate that the herein developed σ₂ receptor ligand [¹⁸F]RM273 has potential to assess by non-invasive molecular imaging the correlation between the availability of σ₂ receptors and properties of brain tumors such as tumor proliferation or resistance towards particular therapies.     

The essential oil fromTagetes minuta L. modulates the binding of [3H]flunitrazepam to crude membranes from chick brain

The hypothesis that the essential oil fromTagetes minuta L. can interact with biological membranes was investigated by assessing its ability of perturbing the binding of a benzodiazepine [flunitrazepam (FNTZ)] to crude membranes from chick brains. The essential oil fromT. minuta L. inhibited [³H]FNTZ specific binding to chick brain membranes. These values were obtained from the analysis of the saturation curve for the kinetic parameters: dissociation equilibrium constant (K_d)=2.47±0.32 nM, maximal binding (B_max)=556±5 fmoles/mg protein, and Hill coefficient (n)=1.00±0.07 in the absence, and K_d=6.73±1.4 nM, B_max=583±69 fmoles/mg protein, and n=1.02±0.08 in the presence of 29 μg/mL of essential oil. The essential oil could self-aggregate with a critical micellar concentration (CMC) of 60 μg/mL. The marked increase in [³H]FNTZ nonspecific binding starting at 60 μg of essence per mL was due to that phenomenon and revealed the ability of self-aggregated structures to interact with membranes. [³H]FNTZ specific binding decrement as a function of essence concentration cannot be ascribed merely to oil's micelles ability of trapping the lipophilic radioligand molecules, because the discontinuous behavior that characterizes a monomer-aggregate phase transition was not shown. Oil's components might behave as competitive inhibitors or allosteric modulators of FNTZ specific binding. However, their ability to increase FNTZ nonspecific binding at concentrations below oil's CMC suggests that this effect may be due to oil's partitioning into the lipid bilayer. This latter phenomenon would induce an increment in membrane fluidity and a change on FNTZ binding site toward a lower affinity conformation. Therefore, the essential oil components can interact with brain membranes either as monomers, by partitioning into the lipid bilayer, or as self-aggregated structures, through an adsorption to the membrane surface.     

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3

The CXCR3 receptor, a class A G protein‐coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure‐based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small‐molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N‐[({1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐2‐[4‐fluoro‐3‐(trifluoromethyl)phenyl]‐N‐{1‐[4‐(3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzyl}piperidin‐4‐yl)methyl]acetamide ( 10 ) showed significant labeling of the CXCR3 receptor (80 %) in a [³H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.     

Co3O4 Nanoparticles Uniformly Dispersed in Rational Porous Carbon Nano-Boxes for Significantly Enhanced Electrocatalytic Detection of H2O2 Released from Living Cells

A facile and ingenious method to chemical etching-coordinating a metal-organic framework (MOF) followed by an annealing treatment was proposed to prepare Co₃O₄ nanoparticles uniformly dispersed in rational porous carbon nano-boxes (Co₃O₄@CNBs), which was further used to detect H₂O₂ released from living cells. The Co₃O₄@CNBs H₂O₂ sensor delivers much higher sensitivity than non-etching/coordinating Co₃O₄, offering a limit of detection of 2.32 nM. The wide working range covers 10 nM-359 μM H₂O₂, while possessing good selectivity and excellent reproducibility. Moreover, this biosensor was used to successfully real-time detect H₂O₂ released from living cells, including both healthy and tumor cells. The excellent performance holds great promise for Co₃O₄@CNBs’s applications in electrochemical biomimetic sensing, particularly real-time monitor H₂O₂ released from living cells.     

A novel hydrogen peroxide biosensor based on Au-graphene-HRP-chitosan biocomposites

Graphene was prepared successfully by introducing -SO₃⁻ to separate the individual sheets. TEM, EDS and Raman spectroscopy were utilized to characterize the morphology and composition of graphene oxide and graphene. To construct the H₂O₂ biosensor, graphene and horseradish peroxidase (HRP) were co-immobilized into biocompatible polymer chitosan (CS), then a glassy carbon electrode (GCE) was modified by the biocomposite, followed by electrodeposition of Au nanoparticles on the surface to fabricate Au/graphene/HRP/CS/GCE. Cyclic voltammetry demonstrated that the direct electron transfer of HRP was realized, and the biosensor had an excellent performance in terms of electrocatalytic reduction towards H₂O₂. The biosensor showed high sensitivity and fast response upon the addition of H₂O₂, under the conditions of pH 6.5, potential −0.3 V. The time to reach the stable-state current was less than 3 s, and the linear range to H₂O₂ was from 5 × 10⁻⁶ M to 5.13 × 10⁻³ M with a detection limit of 1.7 × 10⁻⁶ M (S/N = 3). Moreover, the biosensor exhibited good reproducibility and long-term stability.     

Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A₁R (CPA, CCPA) or A_2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, whereas when administered alone, each compound was ineffective as compared to the control group. Interestingly, the pretreatment with nonselective antagonist caffeine or selective A₁R (DPCPX) or A_2AR (ZM241385) antagonists did not modify the GUO-induced anxiolytic-like effect. Finally, binding assay performed in hippocampal membranes showed that [³H]GUO binding became saturable at 100–300 nM, suggesting the existence of a putative GUO binding site. In competition experiments, ADO showed a potency order similar to GUO in displacing [³H]GUO binding, whereas AR selective agonists, CPA and CGS21680, partially displaced [³H]GUO binding, but the sum of the two effects was able to displace [³H]GUO binding to the same extent of ADO alone. Overall, our results strengthen previous data supporting GUO-mediated anxiolytic-like effects, add new evidence that these effects are blocked by A₁R and A_2AR agonists and pave, although they do not elucidate the mechanism of GUO and ADO receptor interaction, for a better characterization of GUO binding sites in ARs.