RESUMO
G-protein-coupled receptors (GPCRs) can initiate unique functional responses depending on the subcellular site of activation. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have concentrated on the biochemical aspect of this regulation. Here we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism. We devise a strategy to rapidly and selectively redistribute receptor-containing endosomes 'on command' in intact cells without perturbing their biochemical composition. Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cyclic AMP (cAMP)-dependent transcriptional signaling with single-cell resolution. With these, we establish that disruption of native endosome positioning inhibits the initiation of the endosome-dependent transcriptional responses. Finally, we demonstrate a prominent mechanistic role of PDE-mediated cAMP hydrolysis and local protein kinase A activity in this process. Our study, therefore, illuminates a new mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially selective receptor signaling.
Assuntos
Endossomos , Transdução de Sinais , Transdução de Sinais/fisiologia , Endossomos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , AMP Cíclico/metabolismo , FosforilaçãoRESUMO
ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated ß-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.
Assuntos
Antineoplásicos/metabolismo , Antagonistas dos Receptores de Dopamina D2/metabolismo , Imidazóis/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Receptores de Dopamina D2/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Células CHO , Cricetinae , Cricetulus , Antagonistas dos Receptores de Dopamina D2/química , Antagonistas dos Receptores de Dopamina D2/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Imidazóis/química , Imidazóis/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Receptores de Dopamina D2/químicaRESUMO
A variety of neural systems are involved in the brain bases of tobacco addiction. Animal models of nicotine addiction have helped identify a variety of interacting neural systems involved in the pathophysiology of tobacco addiction. We and others have found that drug treatments affecting many of those neurotransmitter systems significantly decrease nicotine self-administration. These treatments include dopamine D1 receptor antagonist, histamine H1 antagonist, serotonin 5HT2C agonist, glutamate NMDA antagonist, nicotinic cholinergic α4ß2 partial agonist and nicotinic cholinergic α3ß4 antagonist acting drugs. It may be the case that combining treatments that affect different neural systems underlying addiction may be more efficacious than single drug treatment. In the current study, we tested the interactions of the D1 antagonist SCH-23390 and the serotonin 5HT2c agonist lorcaserin, both of which we have previously shown to significantly reduce nicotine self-administration. In the acute interactions study, both SCH-23390 and lorcaserin significantly reduced nicotine self-administration when given alone and had additive effects when given in combination. In the chronic study, each drug alone caused a significant decrease in nicotine self-administration. No additive effect was seen in combination because SCH-23390 given alone chronically was already highly effective. Chronic administration of the combination was not seen to significantly prolong reduced nicotine self-administration into the post-treatment period. This research shows that unlike lorcaserin and SCH-23390 interactions when given acutely, when given chronically in combination they do not potentiate or prolong each other's effects in reducing nicotine self-administration.
Assuntos
Nicotina/administração & dosagem , Receptor 5-HT2C de Serotonina/fisiologia , Receptores de Dopamina D1/fisiologia , Autoadministração , Animais , Benzazepinas/farmacologia , Feminino , Ratos , Ratos Sprague-Dawley , Tabagismo/reabilitaçãoRESUMO
Tobacco addiction each year causes millions of deaths worldwide. Brain nicotinic acetylcholine receptors have been shown to be central to tobacco addiction. Nicotine replacement therapy aids tobacco cessation, but the success rate is still far too low. This may in part be due to the fact that neurons with nicotinic receptors are not the only neural systems involved in tobacco addiction. Interacting neural systems also play important roles in tobacco addiction. Nicotine increases the release of a variety of neurotransmitters, including dopamine and serotonin. Dopamine, in particular dopamine D1 receptors, has been shown to be involved in the reinforcing action of nicotine. Serotonin through its actions on 5-HT2C receptors has been shown to play a key role in modulating the reinforcement of addictive drugs, including nicotine and alcohol. Combination of treatments could provide greater treatment efficacy. These studies were conducted to evaluate combination therapies utilizing nicotine replacement therapy in conjunction with either a dopamine D1 receptor antagonist SCH-23390 or a serotonin 5-HT2C receptor agonist, lorcaserin. Female Sprague-Dawley rats were given access to self-administer nicotine via IV infusions. Osmotic pumps were implanted to reproduce the kinetic of chronic nicotine patch therapy. SCH-23390 (0.02â¯mg/kg) or lorcaserin (0.6â¯mg/kg) were administered prior to nicotine self-administration sessions. Reproducing earlier findings SCH-23390, lorcaserin and nicotine replacement therapy were effective at reducing IV nicotine self-administration. 5HT2C agonist treatment had additive effects with chronic nicotine infusion for significantly lowering nicotine self-administration. This study demonstrates the feasibility of combination of chronic nicotine with therapies targeting non-nicotinic receptors as treatment options for tobacco addiction.
Assuntos
Benzazepinas/farmacologia , Benzazepinas/uso terapêutico , Nicotina/administração & dosagem , Receptores de Dopamina D1/antagonistas & inibidores , Agonistas do Receptor 5-HT2 de Serotonina/uso terapêutico , Tabagismo/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Benzazepinas/administração & dosagem , Quimioterapia Combinada , Feminino , Bombas de Infusão Implantáveis , Nicotina/farmacologia , Ratos , Ratos Sprague-Dawley , Autoadministração , Agonistas do Receptor 5-HT2 de Serotonina/administração & dosagem , Abandono do Hábito de Fumar/métodos , Dispositivos para o Abandono do Uso de TabacoRESUMO
The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and ß-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both ß-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.