RESUMO
The yellow fever mosquito, Aedes aegypti, vectors disease-causing agents that adversely affect human health, most notably the viruses causing dengue and yellow fever. The efficacy of current mosquito control programs is challenged by the emergence of insecticide-resistant mosquito populations, suggesting an urgent need for the development of chemical insecticides with new mechanisms of action. One recently identified potential insecticide target is the A. aegypti D1-like dopamine receptor, AaDOP2. The focus of the present study was to evaluate AaDOP2 antagonism both in vitro and in vivo using assay technologies with increased throughput. The in vitro assays revealed AaDOP2 antagonism by four distinct chemical scaffolds from tricyclic antidepressant or antipsychotic chemical classes, and elucidated several structure-activity relationship trends that contributed to enhanced antagonist potency, including lipophilicity, halide substitution on the tricyclic core, and conformational rigidity. Six compounds displayed previously unparalleled potency for in vitro AaDOP2 antagonism, and among these, asenapine, methiothepin, and cis-(Z)-flupenthixol displayed subnanomolar IC50 values and caused rapid toxicity to A. aegypti larvae and/or adults in vivo. Our study revealed a significant correlation between in vitro potency for AaDOP2 antagonism and in vivo toxicity, suggesting viability of AaDOP2 as an insecticidal target. Taken together, this study expanded the repertoire of known AaDOP2 antagonists, enhanced our understanding of AaDOP2 pharmacology, provided further support for rational targeting of AaDOP2, and demonstrated the utility of efficiency-enhancing in vitro and in vivo assay technologies within our genome-to-lead pipeline for the discovery of next-generation insecticides.
Assuntos
Aedes , Antidepressivos , Antipsicóticos , Antagonistas de Dopamina , Proteínas de Insetos/antagonistas & inibidores , Controle de Mosquitos/métodos , Receptores Dopaminérgicos/metabolismo , Aedes/fisiologia , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Larva , Bibliotecas de Moléculas Pequenas , Febre Amarela/transmissãoRESUMO
A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and ß-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate ß-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit ß-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated ß-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate ß-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate ß-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.
Assuntos
Arrestinas/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Arrestinas/metabolismo , Células CHO , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , beta-ArrestinasRESUMO
The D(1) dopamine receptor (D(1)R) has been proposed to form a hetero-oligomer with the D(2) dopamine receptor (D(2)R), which in turn results in a complex that couples to phospholipase C-mediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose-response curves assaying intracellular calcium mobilization in cells heterologously expressing the D(1) and D(2) subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D(1)-D(2) heteromer-selective ligand, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol (SKF83959), found no stimulation of calcium release, but it did find a broad range of cross-reactivity with other G protein-coupled receptors. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Overexpression of G(qα) with the D(1) and D(2) dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G(qα) with only the D1R. Inactivation of Gi or Gs with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D(1)R and D(2)R cotransfected cells. Moreover, sequestration of G(ßγ) subunits through overexpression of G protein receptor kinase 2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggest that the mechanism of D(1)R/D(2)R-mediated calcium signaling involves more than receptor-mediated G(q) protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition, SKF83959 may not exhibit selective activation of D(1)-D(2) heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/análogos & derivados , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Dopamina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistasRESUMO
We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity - ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit â³atypicalâ³ antipsychotic activity in humans with significantly fewer side effects than observed with the currently FDA-approved D2R antagonists.
RESUMO
Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein-NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D1- and D2-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms.
Assuntos
Adenilil Ciclases/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Dopamina/metabolismo , Descoberta de Drogas , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacosRESUMO
To understand the structural basis for the Na(+)-sensitivity of ligand binding to dopamine D2-like receptors, using computational analysis in combination with binding assays, we identified interactions critical in propagating the impact of Na(+) on receptor conformations and on the ligand-binding site. Our findings expand the pharmacologically-relevant conformational spectrum of these receptors.
Assuntos
Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Sódio/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Antagonistas de Dopamina/química , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Ligantes , Simulação de Dinâmica Molecular , Estrutura Molecular , Conformação Proteica/efeitos dos fármacos , Receptores de Dopamina D2/química , Receptores de Dopamina D3/antagonistas & inibidores , Receptores de Dopamina D3/química , Salicilamidas/química , Salicilamidas/farmacologia , Sódio/química , Relação Estrutura-Atividade , Sulpirida/química , Sulpirida/farmacologiaRESUMO
BACKGROUND: New mode-of-action insecticides are sought to provide continued control of pesticide resistant arthropod vectors of neglected tropical diseases (NTDs). We previously identified antagonists of the AaDOP2 D1-like dopamine receptor (DAR) from the yellow fever mosquito, Aedes aegypti, with toxicity to Ae. aegypti larvae as leads for novel insecticides. To extend DAR-based insecticide discovery, we evaluated the molecular and pharmacological characteristics of an orthologous DAR target, CqDOP2, from Culex quinquefasciatus, the vector of lymphatic filariasis and West Nile virus. METHODS/RESULTS: CqDOP2 has 94.7% amino acid identity to AaDOP2 and 28.3% identity to the human D1-like DAR, hD1. CqDOP2 and AaDOP2 exhibited similar pharmacological responses to biogenic amines and DAR antagonists in cell-based assays. The antagonists amitriptyline, amperozide, asenapine, chlorpromazine and doxepin were between 35 to 227-fold more selective at inhibiting the response of CqDOP2 and AaDOP2 in comparison to hD1. Antagonists were toxic to both C. quinquefasciatus and Ae. aegypti larvae, with LC50 values ranging from 41 to 208 µM 72 h post-exposure. Orthologous DOP2 receptors identified from the African malaria mosquito, Anopheles gambiae, the sand fly, Phlebotomus papatasi and the tsetse fly, Glossina morsitans, had high sequence similarity to CqDOP2 and AaDOP2. CONCLUSIONS: DAR antagonists represent a putative new insecticide class with activity against C. quinquefasciatus and Ae. aegypti, the two most important mosquito vectors of NTDs. There has been limited change in the sequence and pharmacological properties of the DOP2 DARs of these species since divergence of the tribes Culicini and Aedini. We identified antagonists selective for mosquito versus human DARs and observed a correlation between DAR pharmacology and the in vivo larval toxicity of antagonists. These data demonstrate that sequence similarity can be predictive of target potential. On this basis, we propose expanded insecticide discovery around orthologous DOP2 targets from additional dipteran vectors.
Assuntos
Aedes/efeitos dos fármacos , Anopheles/efeitos dos fármacos , Culex/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Controle de Insetos , Insetos Vetores/efeitos dos fármacos , Inseticidas/farmacologia , Aedes/parasitologia , Aedes/virologia , Animais , Culex/parasitologia , Culex/virologia , Humanos , Insetos Vetores/parasitologia , Insetos Vetores/virologia , Larva/efeitos dos fármacos , Malária/prevenção & controle , Extratos Vegetais/farmacologia , Receptores Dopaminérgicos/metabolismo , Febre Amarela/prevenção & controleRESUMO
RATIONALE: (-)-Stepholidine is a tetrahydroberberine alkaloid that is known to interact with dopamine receptors and has also been proposed as a novel antipsychotic agent. Its suggested novelty lies in the fact that it has been proposed to have D1-like receptor agonist and D2-like receptor antagonist properties. Thus, it might be effective in treating both positive and negative (cognition) symptoms of schizophrenia. However, its activity on specific dopamine receptor subtypes has not been clarified, especially with respect to its ability to activate D1-like receptors. OBJECTIVES: We wished to examine the affinity and functional activity of (-)-stepholidine at each of the human dopamine receptor subtypes expressed in a defined cellular environment. METHODS: D1-D5 dopamine receptors were stably expressed in cell lines and their interactions with (-)-stepholidine were examined using radioligand binding and various functional signaling assays. Radioligand binding assays were also performed using bovine striatal membranes. RESULTS: (-)-Stepholidine exhibited high (nM) affinity for D1 and D5 receptors, somewhat lower (two- to four-fold) affinity for D2 and D3 receptors, and low micromolar affinity for D4 receptors. Functionally, (-)-stepholidine was ineffective in activating G protein-mediated signaling of D1-like and D2 receptors and was also ineffective in stimulating ß-arrestin recruitment to any dopamine receptor subtype. It did, however, antagonize all of these responses. It also antagonized D1-D2 heteromer-mediated Ca(2+) mobilization. Radioligand binding assays of D1-like receptors in brain membranes also indicated that (-)-stepholidine binds to the D1 receptor with antagonist-like properties. CONCLUSIONS: (-)-Stepholidine is a pan-dopamine receptor antagonist and its in vivo effects are largely mediated through dopamine receptor blockade with potential cross-talk to other receptors or signaling proteins.
Assuntos
Arrestinas/metabolismo , Berberina/análogos & derivados , Encéfalo/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Receptores Dopaminérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Berberina/farmacologia , Encéfalo/metabolismo , Células CHO , Cricetulus , Células HEK293 , Humanos , Receptores de Dopamina D5/metabolismo , beta-ArrestinasRESUMO
The D3 dopamine receptor represents an important target in drug addiction in that reducing receptor activity may attenuate the self-administration of drugs and/or disrupt drug or cue-induced relapse. Medicinal chemistry efforts have led to the development of D3 preferring antagonists and partial agonists that are >100-fold selective vs. the closely related D2 receptor, as best exemplified by extended-length 4-phenylpiperazine derivatives. Based on the D3 receptor crystal structure, these molecules are known to dock to two sites on the receptor where the 4-phenylpiperazine moiety binds to the orthosteric site and an extended aryl amide moiety docks to a secondary binding pocket. The bivalent nature of the receptor binding of these compounds is believed to contribute to their D3 selectivity. In this study, we examined if such compounds might also be "bitopic" such that their aryl amide moieties act as allosteric modulators to further enhance the affinities of the full-length molecules for the receptor. First, we deconstructed several extended-length D3-selective ligands into fragments, termed "synthons", representing either orthosteric or secondary aryl amide pharmacophores and investigated their effects on D3 receptor binding and function. The orthosteric synthons were found to inhibit radioligand binding and to antagonize dopamine activation of the D3 receptor, albeit with lower affinities than the full-length compounds. Notably, the aryl amide-based synthons had no effect on the affinities or potencies of the orthosteric synthons, nor did they have any effect on receptor activation by dopamine. Additionally, pharmacological investigation of the full-length D3-selective antagonists revealed that these compounds interacted with the D3 receptor in a purely competitive manner. Our data further support that the 4-phenylpiperazine D3-selective antagonists are bivalent and that their enhanced affinity for the D3 receptor is due to binding at both the orthosteric site as well as a secondary binding pocket. Importantly, however, their interactions at the secondary site do not allosterically modulate their binding to the orthosteric site.