Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor.

The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently marketed antipsychotic drugs, although effective in managing the symptoms of schizophrenia to a certain extent, are not without their repertoire of serious side effects. There is a need for better therapeutics to treat schizophrenia for which understanding the mechanism of action of the current antipsychotic drugs is imperative. With bioluminescence resonance energy transfer (BRET) assays, we trace the signaling signature of six antipsychotic drugs belonging to three generations at the 5-HT2AR for the entire spectrum of signaling pathways activated by serotonin (5-HT). The antipsychotic drugs display previously unidentified pathway preference at the level of the individual Gα subunits and ß-arrestins. In particular, risperidone, clozapine, olanzapine and haloperidol showed G protein-selective inverse agonist activity. In addition, G protein-selective partial agonism was found for aripiprazole and cariprazine. Pathway-specific apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HT-activated pathways. Computational analyses of the pharmacological and structural fingerprints support a mechanistically based clustering that recapitulate the clinical classification (typical/first generation, atypical/second generation, third generation) of the antipsychotic drugs. The study provides a new framework to functionally classify antipsychotics that should represent a useful tool for the identification of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between specific signaling cascades and in the clinical outcomes of the existing drugs.

Conformation- and activation-based BRET sensors differentially report on GPCR-G protein coupling.

G protein-coupled receptors (GPCRs) regulate cellular signaling processes by coupling to diverse combinations of heterotrimeric G proteins composed of Gα, Gß, and Gγ subunits. Biosensors based on bioluminescence resonance energy transfer (BRET) have advanced our understanding of GPCR functional selectivity. Some BRET biosensors monitor ligand-induced conformational changes in the receptor or G proteins, whereas others monitor the recruitment of downstream effectors to sites of G protein activation. Here, we compared the ability of conformation-and activation-based BRET biosensors to assess the coupling of various class A and B GPCRs to specific Gα proteins in cultured cells. These GPCRs included serotonin 5-HT2A and 5-HT7 receptors, the GLP-1 receptor (GLP-1R), and the M3 muscarinic receptor. We observed different signaling profiles between the two types of sensors, highlighting how data interpretation could be affected by the nature of the biosensor. We also found that the identity of the Gßγ subunits used in the assay could differentially influence the selectivity of a receptor toward Gα subtypes, emphasizing the importance of the receptor-Gßγ pairing in determining Gα coupling specificity. Last, the addition of epitope tags to the receptor could affect stoichiometry and coupling selectivity and yield artifactual findings. These results highlight the need for careful sensor selection and experimental design when probing GPCR-G protein coupling.

G protein-specific mechanisms in the serotonin 5-HT2A receptor regulate psychosis-related effects and memory deficits.

G protein-coupled receptors (GPCRs) are sophisticated signaling machines able to simultaneously elicit multiple intracellular signaling pathways upon activation. Complete (in)activation of all pathways can be counterproductive for specific therapeutic applications. This is the case for the serotonin 2 A receptor (5-HT2AR), a prominent target for the treatment of schizophrenia. In this study, we elucidate the complex 5-HT2AR coupling signature in response to different signaling probes, and its physiological consequences by combining computational modeling, in vitro and in vivo experiments with human postmortem brain studies. We show how chemical modification of the endogenous agonist serotonin dramatically impacts the G protein coupling profile of the 5-HT2AR and the associated behavioral responses. Importantly, among these responses, we demonstrate that memory deficits are regulated by Gαq protein activation, whereas psychosis-related behavior is modulated through Gαi1 stimulation. These findings emphasize the complexity of GPCR pharmacology and physiology and open the path to designing improved therapeutics for the treatment of stchizophrenia.

Enhanced Bystander BRET (ebBRET) Biosensors as Biophysical Tools to Map the Signaling Profile of Neuropsychiatric Drugs Targeting GPCRs.

Bioluminescence resonance energy transfer (BRET) is a non-radiative energy transfer between a bioluminescent donor and a fluorescent acceptor with far-reaching applications in detecting physiologically relevant protein-protein interactions. The recently developed enhanced bystander BRET (ebBRET) biosensors have made it possible to rapidly determine the signaling profile of a series of ligands across a large number of GPCRs and their signaling repertoires, which has tremendous implications in the drug discovery process. Here we describe BRET and the ebBRET biosensors as investigational tools in establishing functional selectivity downstream of GPCRs.

Site-Specific Incorporation of Genetically Encoded Photo-Crosslinkers Locates the Heteromeric Interface of a GPCR Complex in Living Cells.

G protein-coupled receptors (GPCRs) are critical mediators of cell signaling. Although capable of activating G proteins in a monomeric form, numerous studies reveal a possible association of class A GPCRs into dimers/oligomers. The relative location of individual protomers within these GPCR complexes remains a topic of intense debate. We previously reported that class A serotonin 5-HT2A receptor (5-HT2AR) and class C metabotropic glutamate 2 receptor (mGluR2) are able to form a GPCR heterocomplex. By introducing the photoactivatable unnatural amino acid p-azido-L-phenylalanine (azF) at selected individual positions along the transmembrane (TM) segments of mGluR2, we delineate the residues that physically interact at the heteromeric interface of the 5-HT2AR-mGluR2 complex. We show that 5-HT2AR crosslinked with azF incorporated at the intracellular end of mGluR2's TM4, while no crosslinking was observed at other positions along TM1 and TM4. Together, these findings provide important insights into the structural arrangement of the 5-HT2AR-mGluR2 complex.

Revised Pharmacophore Model for 5-HT2A Receptor Antagonists Derived from the Atypical Antipsychotic Agent Risperidone.

Pharmacophore models for 5-HT2A receptor antagonists consist of two aromatic/hydrophobic regions at a given distance from a basic amine. We have previously shown that both aromatic/hydrophobic moieties are unnecessary for binding or antagonist action. Here, we deconstructed the 5-HT2A receptor antagonist/serotonin-dopamine antipsychotic agent risperidone into smaller structural segments that were tested for 5-HT2A receptor affinity and function. We show, again, that the entire risperidone structure is unnecessary for retention of affinity or antagonist action. Replacement of the 6-fluoro-3-(4-piperidinyl)-1,2-benz[ d]isoxazole moiety by isosteric tryptamines resulted in retention of affinity and antagonist action. Additionally, 3-(4-piperidinyl)-1,2-benz[ d]isoxazole (10), which represents less than half the structural features of risperidone, retains both affinity and antagonist actions. 5-HT2A receptor homology modeling/docking studies suggest that 10 binds in a manner similar to risperidone and that there is a large cavity to accept various N4-substituted analogues of 10 such as risperidone and related agents. Alterations of this "extended" moiety improve receptor binding and functional potency. We propose a new risperidone-based pharmacophore for 5-HT2A receptor antagonist action.

Contribution of heteromerization to G protein-coupled receptor function.

G protein-coupled receptors (GPCRs) are a remarkably multifaceted family of transmembrane proteins that exert a variety of physiological effects. Although family A GPCRs are able to operate as monomers, there is increasing evidence that heteromerization represents a fundamental aspect of receptor function, trafficking and pharmacology. Most recently, it has been suggested that GPCR heteromers may play a crucial role as new molecular targets of heteromer-selective and bivalent ligands. The current review summarizes key recent developments in these topics.

Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects.

Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT2A-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment.

Reformulating a Pharmacophore for 5-HT2A Serotonin Receptor Antagonists.

Several pharmacophore models have been proposed for 5-HT2A serotonin receptor antagonists. These typically consist of two aromatic/hydrophobic moieties separated by a given distance from each other, and from a basic amine. Although specified distances might vary, the models are relatively similar in their general construction. Because our preliminary data indicated that two aromatic (hydrophobic) moieties might not be required for such action, we deconstructed the serotonin-dopamine antipsychotic agent risperidone (1) into four smaller structural fragments that were thoroughly examined in 5-HT2A receptor binding and functional (i.e., two-electrode voltage clamp (TEVC) and intracellular calcium release) assays. It was apparent that truncated risperidone analogues behaved as antagonists. In particular, 6-fluoro-3-(1-methylpiperidin-4-yl)benzisoxazole (4) displayed high affinity for 5-HT2A receptors (Ki of ca. 12 nM) relative to risperidone (Ki of ca. 5 nM) and behaved as a potent 5-HT2A serotonin receptor antagonist. These results suggest that multiple aromatic (hydrophobic) moieties are not essential for high-affinity 5-HT2A receptor binding and antagonist activity and that current pharmacophore models for such agents are very much in need of revision.