Ligand bias and inverse agonism on 5-HT2A receptor-mediated modulation of G protein activity in post-mortem human brain.

BACKGROUND AND PURPOSE: Whereas biased agonism on the 5-HT2A receptor has been ascribed to hallucinogenic properties of psychedelics, no information about biased inverse agonism on this receptor is available. In schizophrenia, increased 5-HT2A receptor constitutive activity has been suggested, highlighting the therapeutic relevance of inverse agonism. This study characterized the modulation of G protein activity promoted by different drugs, commonly considered as 5-HT2A receptor antagonists, in post-mortem human brain cortex. EXPERIMENTAL APPROACH: Modulation of [35S]GTPγS binding to different subtypes of Gα proteins exerted by different 5-HT2A receptor drugs was determined by scintillation proximity assays in brain from human, WT and 5-HT2A receptor KO mice. KEY RESULTS: MDL-11,939 was the only drug having no effect on the basal activity of 5-HT2A receptor. Altanserin and pimavanserin decreased basal activation of Gi1, but not Gq/11 proteins. This effect was blocked by MDL-11,939 and absent in 5-HT2A receptor KO mice. Volinanserin showed 5-HT2A receptor-mediated inverse agonism both on Gi1 and Gq/11 proteins. Ketanserin exhibited 5-HT2A receptor partial agonism exclusively on Gq/11 proteins. On the other hand, eplivanserin and nelotanserin displayed inverse agonism on Gq/11 and/or Gi1 proteins, which was insensitive to MDL-11,939 and was present in KO mice suggesting a role for another receptor. CONCLUSION AND IMPLICATIONS: The results reveal the existence of constitutively active 5-HT2A receptors in human pre-frontal cortex and demonstrate different pharmacological profiles of various 5-HT2A receptor drugs previously considered antagonists. These findings indicate that altanserin and pimavanserin possess biased inverse agonist profile towards 5-HT2A receptor activation of Gi1 proteins.

The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.

Effect of antipsychotic drugs on group II metabotropic glutamate receptor expression and epigenetic control in postmortem brains of schizophrenia subjects.

Antipsychotic-induced low availability of group II metabotropic glutamate receptors (including mGlu2R and mGlu3R) in brains of schizophrenia patients may explain the limited efficacy of mGlu2/3R ligands in clinical trials. Studies evaluating mGlu2/3R levels in well-designed, large postmortem brain cohorts are needed to address this issue. Postmortem samples from the dorsolateral prefrontal cortex of 96 schizophrenia subjects and matched controls were collected. Toxicological analyses identified cases who were (AP+) or were not (AP-) receiving antipsychotic treatment near the time of death. Protein and mRNA levels of mGlu2R and mGlu3R, as well as GRM2 and GRM3 promoter-attached histone posttranslational modifications, were quantified. Experimental animal models were used to compare with data obtained in human tissues. Compared to matched controls, schizophrenia cortical samples had lower mGlu2R protein amounts, regardless of antipsychotic medication. Downregulation of mGlu3R was observed in AP- schizophrenia subjects only. Greater predicted occupancy values of dopamine D2 and serotonin 5HT2A receptors correlated with higher density of mGlu3R, but not mGlu2R. Clozapine treatment and maternal immune activation in rodents mimicked the mGlu2R, but not mGlu3R regulation observed in schizophrenia brains. mGlu2R and mGlu3R mRNA levels, and the epigenetic control mechanisms did not parallel the alterations at the protein level, and in some groups correlated inversely. Insufficient cortical availability of mGlu2R and mGlu3R may be associated with schizophrenia. Antipsychotic treatment may normalize mGlu3R, but not mGlu2R protein levels. A model in which epigenetic feedback mechanisms controlling mGlu3R expression are activated to counterbalance mGluR loss of function is described.

Selective inhibition of cannabinoid CB1 receptor-evoked signalling by the interacting protein GAP43.

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB1 receptor (CB1R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB1R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB1R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB1R and GAP43 and show that GAP43 modulates CB1R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB1R/Gi/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB1R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB1R-mediated activation of the cytoskeleton-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB1R/Gq/11 protein coupling. GAP43 also abrogated CB1R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB1R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB1R-Gq/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.

Design, Synthesis, Molecular Docking, and Biological Evaluation of Novel Pimavanserin-Based Analogues as Potential Serotonin 5-HT2A Receptor Inverse Agonists.

There is concern for important adverse effects with use of second-generation antipsychotics in Parkinson's disease psychosis (PDP) and dementia-related psychosis. Pimavanserin is the only antipsychotic drug authorized for PDP and represents an inverse agonist of 5-HT2A receptors (5-HT2AR) lacking affinity for dopamine receptors. Therefore, the development of serotonin 5-HT2AR inverse agonists without dopaminergic activity represents a challenge for different neuropsychiatric disorders. Using ligand-based drug design, we discovered a novel structure of pimavanserin analogues (2, 3, and 4). In vitro competition receptor binding and functional G protein coupling assays demonstrated that compounds 2, 3, and 4 showed higher potency than pimavanserin as 5-HT2AR inverse agonists in the human brain cortex and recombinant cells. To assess the effect of molecular substituents for selectivity and inverse agonism at 5-HT2ARs, molecular docking and in silico predicted physicochemical parameters were performed. Docking studies were in agreement with in vitro screenings and the results resembled pimavanserin.

Isolation and Differentiation of Neurons and Glial Cells from Olfactory Epithelium in Living Subjects.

The study of psychiatric and neurological diseases requires the substrate in which the disorders occur, that is, the nervous tissue. Currently, several types of human bio-specimens are being used for research, including postmortem brains, cerebrospinal fluid, induced pluripotent stem (iPS) cells, and induced neuronal (iN) cells. However, these samples are far from providing a useful predictive, diagnostic, or prognostic biomarker. The olfactory epithelium is a region close to the brain that has received increased interest as a research tool for the study of brain mechanisms in complex neuropsychiatric and neurological diseases. The olfactory sensory neurons are replaced by neurogenesis throughout adult life from stem cells on the basement membrane. These stem cells are multipotent and can be propagated in neurospheres, proliferated in vitro and differentiated into multiple cell types including neurons and glia. For all these reasons, olfactory epithelium provides a unique resource for investigating neuronal molecular markers of neuropsychiatric and neurological diseases. Here, we describe the isolation and culture of human differentiated neurons and glial cells from olfactory epithelium of living subjects by an easy and non-invasive exfoliation method that may serve as a useful tool for the research in brain diseases.

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

α2A- and α2C-adrenoceptor expression and functionality in postmortem prefrontal cortex of schizophrenia subjects.

Previous evidence suggests that α2-adrenoceptors (α2-AR) may be involved in the pathophysiology of schizophrenia. However, postmortem brain studies on α2-AR expression and functionality in schizophrenia are scarce. The aim of our work was to evaluate α2A-AR and α2C-AR expression in different subcellular fractions of prefrontal cortex postmortem tissue from antipsychotic-free (absence of antipsychotics in blood at the time of death) (n = 12) and antipsychotic-treated (n = 12) subjects with schizophrenia, and matched controls (n = 24). Functional coupling of α2-AR to Gα proteins induced by the agonist UK14304 was also tested. Additionally, Gα protein expression was also evaluated. In antipsychotic-free schizophrenia subjects, α2A-AR and α2C-AR protein expression was similar to controls in all the subcellular fractions. Conversely, in antipsychotic-treated schizophrenia subjects, increased α2A-AR expression was found in synaptosomal plasma membrane and postsynaptic density (PSD) fractions (+60% and +79% vs controls, respectively) with no significant changes in α2C-AR. [35S]GTPγS SPA experiments showed a significant lower stimulation of Gαi2 and Gαi3 proteins by UK14304 in antipsychotic-treated schizophrenia subjects, whereas stimulation in antipsychotic-free schizophrenia subjects remained unchanged. Gαo protein stimulation was significantly decreased in both antipsychotic-free and antipsychotic-treated schizophrenia subjects compared to controls. Expression of Gαi3 protein did not differ between groups, whereas Gαi2 levels were increased in PSD of schizophrenia subjects, both antipsychotic-free and antipsychotic-treated. Gαo protein expression was increased in PSD of antipsychotic-treated subjects and in the presynaptic fraction of antipsychotic-free schizophrenia subjects. The present results suggest that antipsychotic treatment is able to modify in opposite directions both the protein expression and the functionality of α2A-AR in the cortex of schizophrenia patients.

Characterization of dopamine D2 receptor coupling to G proteins in postmortem brain of subjects with schizophrenia.

BACKGROUND: Alterations of dopamine D1 (D1R) and D2 receptor (D2R) are proposed in schizophrenia but brain neuroimaging and postmortem studies have shown controversial results in relation to D1R and D2R density. Besides, scarce information on the functionality of brain D1R and D2R is available. The present study characterized G-protein activation by D1R and D2R agonists in postmortem human brain. Furthermore, D2R functional status was compared between schizophrenia and control subjects. METHODS: G-protein receptor coupling was assessed in control caudate nucleus and frontal cortex by [35S]GTPγS-binding stimulation induced by increasing concentrations (10-10-10-3 M) of dopamine, and the selective dopaminergic agonists SKF38393 (D1R) and NPA (D2R). Concentration-response curves to NPA stimulation of [35S]GTPγS binding were analyzed in antipsychotic-free (n = 10) and antipsychotic-treated (n = 7) schizophrenia subjects and matched controls (n = 17). RESULTS: In caudate, [35S]GTPγS-binding responses to agonists were compatible with the existence of functional D2R. In contrast, stimulations in cortex showed responses that did not correspond to D1R or D2R. [35S]GTPγS-binding activation by NPA in caudate displayed biphasic curves with similar profile in schizophrenia (EC50H = 7.94 nM; EC50L = 7.08 µM) and control (EC50H = 7.24 nM; EC50L = 15.14 µM) subjects. The presence or absence of antipsychotic medication did not influence the pharmacological parameters. CONCLUSIONS: Feasibility of functional evaluation of dopamine receptors in postmortem human brain by conventional [35S]GTPγS-binding assays appears to be restricted to signalling through inhibitory Gi/o proteins. These findings provide functional information about brain D2R status in subjects with schizophrenia and do not support the existence of D2R supersensitive in this mental disorder.

Opposite alterations of 5-HT2A receptor brain density in subjects with schizophrenia: relevance of radiotracers pharmacological profile.

The status of serotonin 5-HT2A receptors (5-HT2ARs) in schizophrenia has been controversial. In vivo positron emission tomography neuroimaging and in vitro post-mortem binding studies have reported conflicting results about 5-HT2AR density. Radiotracers bind different receptor conformations depending on their agonist, antagonist or inverse agonist properties. This study investigates 5-HT2AR density in the post-mortem prefrontal cortex from subjects with schizophrenia and controls using three radiotracers with a different pharmacological profile. The specific binding parameters of the inverse agonist [18F]altanserin, the agonist [3H]lysergic acid diethylamide (LSD) and the antagonist [3H]MDL100907 to brain cortex membranes from 20 subjects with schizophrenia and 20 individually matched controls were evaluated under similar methodological conditions. Ten schizophrenia subjects were antipsychotic-free at death. Saturation curve analyses were performed by non-linear regression to obtain a maximal density of binding sites (Bmax) and the affinity of the respective radiotracers (Kd). In schizophrenia subjects, 5-HT2AR density was decreased when quantified by [18F]altanserin binding, whereas increased when evaluated by [3H]LSD binding. However, [3H]MDL100907 binding was unaltered. A slight loss of affinity (higher Kd) was observed exclusively in [3H]LSD binding. The findings were more evident in antipsychotic-free subjects than in antipsychotic-treated subjects. In conclusion, a higher proportion of the 5-HT2AR-active functional conformation, which is rather identified by agonist radiotracers, was observed in schizophrenia patients. A consequent reduction of the inactive 5-HT2AR conformation, which is preferentially identified by inverse agonist radiotracers, was also obtained. Antagonist radiotracers do not distinguish between molecular conformations of the receptor, and accordingly, the absence of changes was shown. These results are compatible with the proposed increased functional activity of brain cortical 5-HT2ARs in schizophrenia.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

Serotonin 2A receptors and cannabinoids.

Accumulating evidence has proven that both exogenous cannabinoids as well as imbalances in the endocannabinoid system are involved in the onset and development of mental disorders such as anxiety, depression, or schizophrenia. Extensive recent research in this topic has mainly focused on the molecular mechanisms by which cannabinoid agonists may contribute to the pathophysiology of these disorders. Initially, serotonin neurotransmitter garnered most attention due to its relationship to mood disorders and mental diseases, with little attention to specific receptors. To date, the focus has redirected toward the understanding of different serotonin receptors, through a demonstration of its versatile pharmacology and synergy with different modulators. Serotonin 2A receptors are a good example of this phenomenon, and the complex signaling that they trigger appears of high relevance in the context of mental disorders, especially in schizophrenia. This chapter will analyze most relevant attributes of serotonin 2A receptors and the endocannabinoid system, and will highlight the evidence toward the functional bidirectional interaction between these elements in the brain as well as the impact of the endocannabinoid system dysregulation on serotonin 2A receptors functionality.

Adrenergic Modulation With Photochromic Ligands.

Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.

Pimavanserin exhibits serotonin 5-HT2A receptor inverse agonism for Gαi1- and neutral antagonism for Gαq/11-proteins in human brain cortex.

Pimavanserin is claimed as the first antipsychotic drug that shows selectivity for serotonin 5-HT2 receptors (5-HT2Rs) and lacks of affinity for dopamine D2 receptors (D2Rs). Cell-based functional assays suggest that pimavanserin and antipsychotics with D2R/5-HT2R affinity could act as inverse agonists of 5-HT2ARs. However, there is not evidence of such pharmacological profile in native brain tissue. 5-HT2ARs are able to engage both canonical Gαq/11- and non-canonical Gαi1-proteins. In the present study, the response to pimavanserin of the 5-HT2AR coupling to Gαq/11- and Gαi1-proteins was measured in membranes of postmortem human prefrontal cortex by antibody-capture [35S]GTPγS binding scintillation proximity assays. Pimavanserin promoted a concentration-dependant inhibition of the 5-HT2AR coupling to Gαi1-proteins whereas the response of Gαq/11-proteins was unaltered, suggesting inverse agonism and neutral antagonism properties, respectively. The inhibition was abolished in the presence of the selective 5-HT2AR antagonist MDL-11,939 and was absent in brain cortex of 5-HT2AR knock-out mice when compared to respective 5-HT2AR wild-type animals. In conclusion, the results demonstrate the existence of constitutive 5-HT2AR activity in human brain for the signalling pathway mediated by Gαi1-proteins. Pimavanserin demonstrates 5-HT2AR functional selectivity and exhibits inverse agonist profile towards Gαi1-proteins, which is considered the effector pathway promoting hallucinogenic responses. In contrast, pimavanserin behaves as neutral antagonist on the 5-HT2AR coupling to the canonical Gαq/11-protein pathway. The results strengthen the relevance of inverse agonism as potential mechanism of antipsychotic activity. Moreover, the existence of functional selectivity of 5-HT2ARs for different Gα-proteins could contribute to better design of 5-HT2AR-related antipsychotic drugs.

Ribosomal Protein S6 Hypofunction in Postmortem Human Brain Links mTORC1-Dependent Signaling and Schizophrenia.

The mechanistic target of rapamycin (also known as mammalian target of rapamycin) (mTOR)-dependent signaling pathway plays an important role in protein synthesis, cell growth, and proliferation, and has been linked to the development of the central nervous system. Recent studies suggest that mTOR signaling pathway dysfunction could be involved in the etiopathogenesis of schizophrenia. The main goal of this study was to evaluate the status of mTOR signaling pathway in postmortem prefrontal cortex (PFC) samples of subjects with schizophrenia. For this purpose, we quantified the protein expression and phosphorylation status of the mTOR downstream effector ribosomal protein S6 as well as other pathway interactors such as Akt and GSK3ß. Furthermore, we quantified the status of these proteins in the brain cortex of rats chronically treated with the antipsychotics haloperidol, clozapine, or risperidone. We found a striking decrease in the expression of total S6 and in its active phosphorylated form phospho-S6 (Ser235/236) in the brain of subjects with schizophrenia compared to matched controls. The chronic treatment with the antipsychotics haloperidol and clozapine affected both the expression of GSK3ß and the activation of Akt [phospho-Akt (Ser473)] in rat brain cortex, while no changes were observed in S6 and phospho-S6 (Ser235/236) protein expression with any antipsychotic treatment. These findings provide further evidence for the involvement of the mTOR-dependent signaling pathway in schizophrenia and suggest that a hypofunctional S6 may have a role in the etiopathogenesis of this disorder.

Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins.

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30-40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Big Data Challenges Targeting Proteins in GPCR Signaling Pathways; Combining PTML-ChEMBL Models and [35S]GTPγS Binding Assays.

G-protein-coupled receptors (GPCRs), also known as 7-transmembrane receptors, are the single largest class of drug targets. Consequently, a large amount of preclinical assays having GPCRs as molecular targets has been released to public sources like the Chemical European Molecular Biology Laboratory (ChEMBL) database. These data are also very complex covering changes in drug chemical structure and assay conditions like c0 = activity parameter (Ki, IC50, etc.), c1 = target protein, c2 = cell line, c3 = assay organism, etc., making difficult the analysis of these databases that are placed in the borders of a Big Data challenge. One of the aims of this work is to develop a computational model able to predict new GPCRs targeting drugs taking into consideration multiple conditions of assay. Another objective is to perform new predictive and experimental studies of selective 5-HTA2 receptor agonist, antagonist, or inverse agonist in human comparing the results with those from the literature. In this work, we combined Perturbation Theory (PT) and Machine Learning (ML) to seek a general PTML model for this data set. We analyzed 343 738 unique compounds with 812 072 end points (assay outcomes), with 185 different experimental parameters, 592 protein targets, 51 cell lines, and/or 55 organisms (species). The best PTML linear model found has three input variables only and predicted 56 202/58 653 positive outcomes (sensitivity = 95.8%) and 470 230/550 401 control cases (specificity = 85.4%) in training series. The model also predicted correctly 18 732/19 549 (95.8%) of positive outcomes and 156 739/183 469 (85.4%) of cases in external validation series. To illustrate its practical use, we used the model to predict the outcomes of six different 5-HT2A receptor drugs, namely, TCB-2, DOI, DOB, altanserin, pimavanserin, and nelotanserin, in a very large number of different pharmacological assays. 5-HT2A receptors are altered in schizophrenia and represent drug target for antipsychotic therapeutic activity. The model correctly predicted 93.83% (76 of 86) experimental results for these compounds reported in ChEMBL. Moreover, [35S]GTPγS binding assays were performed experimentally with the same six drugs with the aim of determining their potency and efficacy in the modulation of G-proteins in human brain tissue. The antagonist ketanserin was included as inactive drug with demonstrated affinity for 5-HT2A/C receptors. Our results demonstrate that some of these drugs, previously described as serotonin 5-HT2A receptor agonists, antagonists, or inverse agonists, are not so specific and show different intrinsic activity to that previously reported. Overall, this work opens a new gate for the prediction of GPCRs targeting compounds.

Chronic fluoxetine reverses the effects of chronic corticosterone treatment on α2-adrenoceptors in the rat frontal cortex but not locus coeruleus.

Disruption of the hypothalamic-pituitary-adrenal axis is an established finding in patients with anxiety and/or depression. Chronic corticosterone administration in animals has been proposed as a model for the study of these stress-related disorders and the antidepressant action. Alterations of the central noradrenergic system and specifically of inhibitory α2-adrenoceptors seem to be part of the pathophysiology of depression and contribute to the antidepressant activity. The present study evaluates in male rats the effect of chronic corticosterone treatment during 35 days (16-20 mg kg-1 day-1) on the sensitivity of α2-adrenoceptors expressed in the somatodendritic and terminal noradrenergic areas locus coeruleus (LC) and prefrontal cortex (PFC), respectively. Further, the effect of chronic fluoxetine treatment (5 mg kg-1, i.p., since the 15th day) on the sensitivity of α2-adrenoceptors was examined under control conditions and in corticosterone-treated rats. The α2-adrenoceptor functionality was analysed in vitro by agonist-mediated [35S]GTPγS binding stimulation and in vivo through the modulation of noradrenaline (NA) release evaluated by dual-probe microdialysis. The concentration-effect curves of the [35S]GTPγS binding stimulation by the agonist UK14304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) demonstrated a desensitization of cortical α2-adrenoceptors induced by corticosterone (-logEC50 = 6.7 ±â€¯0.2 vs 8.2 ±â€¯0.3 in controls) that was reverted by fluoxetine treatment (-logEC50 = 7.5 ±â€¯0.3). Local administration of the α2-adrenoceptor antagonist RS79948 ((8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) (0.1-100 µmol L-1) into the LC induced a concentration-dependent NA increase in the PFC of the control group (Emax = 191 ±â€¯30%) but non-significant effect was observed in corticosterone-treated rats (Emax = 133 ±â€¯46%), reflecting a desensitization of α2-adrenoceptors that control the firing of noradrenergic neurons. Fluoxetine treatment did not alter the corticosterone-induced desensitization in this area (Emax = 136 ±â€¯19%). No effect of fluoxetine on α2-adrenoceptor functionality was observed in control animals (Emax = 223 ±â€¯30%). In PFC, the local administration of RS79948 increased NA in controls (Emax = 226 ±â€¯27%) without effect in the corticosterone group (Emax = 115 ±â€¯26%), suggesting a corticosterone-induced desensitization of terminal α2-adrenoceptors. Fluoxetine administration prevented the desensitization induced by corticosterone in the PFC (Emax = 233 ±â€¯33%) whereas desensitized α2-adrenoceptors in control animals (Emax = -24 ±â€¯10%). These data indicate that chronic corticosterone increases noradrenergic activity by acting at different α2-adrenoceptor subpopulations. Treatment with the antidepressant fluoxetine seems to counteract these changes by acting mainly on presynaptic α2-adrenoceptors expressed in terminal areas.

Therapeutic targeting of HER2-CB2R heteromers in HER2-positive breast cancer.

Although human epidermal growth factor receptor 2 (HER2)-targeted therapies have dramatically improved the clinical outcome of HER2-positive breast cancer patients, innate and acquired resistance remains an important clinical challenge. New therapeutic approaches and diagnostic tools for identification, stratification, and treatment of patients at higher risk of resistance and recurrence are therefore warranted. Here, we unveil a mechanism controlling the oncogenic activity of HER2: heteromerization with the cannabinoid receptor CB2R. We show that HER2 physically interacts with CB2R in breast cancer cells, and that the expression of these heteromers correlates with poor patient prognosis. The cannabinoid Δ9-tetrahydrocannabinol (THC) disrupts HER2-CB2R complexes by selectively binding to CB2R, which leads to (i) the inactivation of HER2 through disruption of HER2-HER2 homodimers, and (ii) the subsequent degradation of HER2 by the proteasome via the E3 ligase c-CBL. This in turn triggers antitumor responses in vitro and in vivo. Selective targeting of CB2R transmembrane region 5 mimicked THC effects. Together, these findings define HER2-CB2R heteromers as new potential targets for antitumor therapies and biomarkers with prognostic value in HER2-positive breast cancer.