RESUMO
Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a significant step in receptor kinetics and is known to be involved in receptor down-regulation. However, the dopamine D3 receptor (D3R) has been an exception wherein agonist induces D3Rs to undergo desensitization followed by pharmacological sequestration - which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization. Pharmacological sequestration renders the receptor in an inactive state on the membrane. In our previous study we demonstrated that a novel class of D3R agonists exemplified by SK608 have biased signaling properties via the G-protein dependent pathway and do not induce D3R desensitization. In this study, using radioligand binding assay, immunoblot or immunocytochemistry methods, we observed that SK608 induced internalization of human D3R stably expressed in CHO, HEK and SH-SY5Y cells which are derived from neuroblastoma cells, suggesting that it is not a cell-type specific event. Further, we have evaluated the potential mechanism of D3R internalization induced by these biased signaling agonists. SK608-induced D3R internalization was time- and concentration-dependent. In comparison, dopamine induced D3R upregulation and pharmacological sequestration in the same assays. GRK2 and clathrin/dynamin I/II are the key molecular players in the SK608-induced D3R internalization process, while ß-arrestin 1/2 and GRK-interacting protein 1(GIT1) are not involved. These results suggest that SK608-promoted D3R internalization is similar to the type II internalization observed among peptide binding GPCRs.
Assuntos
Butilaminas/farmacologia , Agonistas de Dopamina/farmacologia , Receptores de Dopamina D3/agonistas , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Dopamina/farmacologia , Células HEK293 , Humanos , Transporte Proteico/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Transdução de Sinais , beta-Arrestinas/genética , beta-Arrestinas/metabolismoRESUMO
We have developed a series of carbazole-derived compounds based on our hybrid D2/D3 agonist template to design multifunctional compounds for the symptomatic and disease-modifying treatment of Parkinson's disease (PD). The lead molecules (-)-11b (D-636), (-)-15a (D-653), and (-)-15c (D-656) exhibited high affinity for both D2 and D3 receptors and in GTPγS functional assay, the compounds showed potent agonist activity at both D2 and D3 receptors (EC50 (GTPγS); D2 = 48.7 nM, D3 = 0.96 nM for 11b, D2 = 0.87 nM, D3 = 0.23 nM for 15a and D2 = 2.29 nM, D3 = 0.22 nM for 15c). In an animal model of PD, the test compounds exhibited potent in vivo activity in reversing hypolocomotion in reserpinized rats with a long duration of action compared to the reference drug ropinirole. In a cellular antioxidant assay, compounds (-)-11b, (-)-15a, and (-)-15c exhibited potent activity in reducing oxidative stress induced by neurotoxin 6-hydroxydopamine (6-OHDA). Also, in a cell-based PD neuroprotection model, these lead compounds significantly increased cell survival from toxicity of 6-OHDA, thereby producing a neuroprotective effect. Additionally, compounds (-)-11b and (-)-15a inhibited aggregation and reduced toxicity of recombinant alpha synuclein protein in a cell based in vitro assay. These observations suggest that the lead carbazole-based dopamine agonists may be promising multifunctional molecules for a viable symptomatic and disease-modifying therapy of PD and should be further investigated.
Assuntos
Carbazóis/síntese química , Agonistas de Dopamina/síntese química , Desenho de Fármacos , Doença de Parkinson/metabolismo , Animais , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Agonistas de Dopamina/farmacologia , Agonistas de Dopamina/uso terapêutico , Feminino , Masculino , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Oxidopamina/toxicidade , Células PC12 , Doença de Parkinson/tratamento farmacológico , Ratos , Ratos Sprague-DawleyRESUMO
The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 µM) and amphetamine-stimulated efflux (59% reduction at 10 µM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Dopamina/metabolismo , Tamoxifeno/farmacologia , Anfetamina/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/análogos & derivados , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Masculino , Ratos , Suínos , Sinaptossomos/metabolismoRESUMO
Parkinson's disease (PD) is a progressive neurodegenerative disorder, and development of disease-modifying treatment is still an unmet medical need. Considering the implication of free iron(II) in PD, we report here the design and characterization of a novel hybrid iron chelator, (-)-12 (D-607) as a multitarget-directed ligand against PD. Binding and functional assays at dopamine D2/D3 receptors indicate potent agonist activity of (-)-12. The molecule displayed an efficient preferential iron(II) chelation properties along with potent in vivo activity in a reserpinized PD animal model. The compound also rescued PC12 cells from toxicity induced by iron delivered intracellularly in a dose-dependent manner. However, Fe3+ selective dopamine agonist 1 and a well-known antiparkinsonian drug pramipexole produced little to no neuroprotection effect under the same experimental condition. These observations strongly suggest that (-)-12 should be a promising multifunctional lead molecule for a viable symptomatic and disease modifying therapy of PD.
Assuntos
2,2'-Dipiridil/análogos & derivados , Antiparkinsonianos/farmacologia , Agonistas de Dopamina/farmacologia , Quelantes de Ferro/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson , Piperazinas/farmacologia , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Animais , Antiparkinsonianos/síntese química , Antiparkinsonianos/química , Modelos Animais de Doenças , Agonistas de Dopamina/síntese química , Agonistas de Dopamina/química , Quelantes de Ferro/síntese química , Quelantes de Ferro/química , Camundongos , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Células PC12 , Piperazinas/síntese química , Piperazinas/química , RatosRESUMO
Our ongoing drug development endeavor to design compounds for symptomatic and neuroprotective treatment of Parkinson's disease (PD) led us to carry out a structure activity relationship study based on dopamine agonists pramipexole and 5-OHDPAT. Our goal was to incorporate structural elements in these agonists in a way to preserve their agonist activity while producing inhibitory activity against aggregation of α-synuclein protein. In our design we appended various catechol and related phenol derivatives to the parent agonists via different linker lengths. Structural optimization led to development of several potent agonists among which (-)-8a, (-)-14 and (-)-20 exhibited potent neuroprotective properties in a cellular PD model involving neurotoxin 6-OHDA. The lead compounds (-)-8a and (-)-14 were able to modulate aggregation of α-synuclein protein efficiently. Finally, in an in vivo PD animal model, compound (-)-8a exhibited efficacious anti-parkinsonian effect.
Assuntos
Agonistas de Dopamina/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D3/agonistas , alfa-Sinucleína/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Agonistas de Dopamina/síntese química , Agonistas de Dopamina/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Oxidopamina/toxicidade , Células PC12 , Agregados Proteicos/efeitos dos fármacos , Ratos , Relação Estrutura-Atividade , alfa-Sinucleína/metabolismoRESUMO
We have developed a series of dihydroxy compounds and related analogues based on our hybrid D2/D3 agonist molecular template to develop multifunctional drugs for symptomatic and neuroprotective treatment for Parkinson's disease (PD). The lead compound (-)-24b (D-520) exhibited high agonist potency at D2/D3 receptors and produced efficacious activity in the animal models for PD. The data from thioflavin T (ThT) assay and from transmission electron microscopy (TEM) analysis demonstrate that D-520 is able to modulate aggregation of alpha-synuclein (αSN). Additionally, coincubation of D-520 with αSN is able to reduce toxicity of preformed aggregates of αSN compared to control αSN alone. Finally, in a neuroprotection study with dopaminergic MN9D cells, D-520 clearly demonstrated the effect of neuroprotection from toxicity of 6-hydroxydopamine. Thus, compound D-520 possesses properties characteristic of multifunctionality conducive to symptomatic and neuroprotective treatment of PD.
Assuntos
Antiparkinsonianos/farmacologia , Agonistas de Dopamina/farmacologia , Fármacos Neuroprotetores/farmacologia , Receptores de Dopamina D3/agonistas , alfa-Sinucleína/metabolismo , Animais , Antiparkinsonianos/síntese química , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Agonistas de Dopamina/síntese química , Neurônios Dopaminérgicos/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Oxidopamina , Transtornos Parkinsonianos/tratamento farmacológico , Ratos Sprague-Dawley , ReserpinaRESUMO
The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET.
Assuntos
Cisteína/metabolismo , Histidina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , HumanosRESUMO
Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d-amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co-immunoprecipitation and cross-linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild-type (WT) DAT with W84L mutant, effects of d-amphetamine on oligomerization (decrease) but not surface DAT are observed. Internalization of W84L monomers appears to be slow as inferred from the inability of d-amphetamine to reduce surface Myc upon co-expressing Flag-WT with Myc-W84L but not Myc-WT with Flag-W84L, and from the sluggish Myc-W84L endocytosis rate (both with or without d-amphetamine). Results obtained for D313N, D345N, or D436N mutants can all be accommodated by a model in which D-amphetamine is unable to dissociate mutant protomers from oligomers (tetramers or higher-order assemblies) that contain them; this interpretation is confirmed in experiments with both tag reversal in co-expression and antibody reversal in western blotting. Upon co-transfecting Myc- and Flag-tagged constructs, resulting tetramers can be calculated to be composed of different species (MycMycMycMyc, MycMycMycFlag, MycMycFlagFlag, MycFlagFlagFlag, and FlagFlagFlagFlag), but it is shown that outcomes predicted by models based on MycMycFlagFlag oligomers are not changed in a major way by the occurrence of the additional species.
Assuntos
Anfetamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/química , Modelos Moleculares , Modelos Neurológicos , Mutação , Anfetamina/farmacologia , Linhagem Celular , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Mutação/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Propriedades de SuperfícieRESUMO
The role of iron in the pathogenesis of Parkinson's disease (PD) has been implicated strongly because of generation of oxidative stress leading to dopamine cell death. In our overall goal to develop bifunctional/multifunctional drugs, we designed dopamine D2/D3 agonist molecules with a capacity to bind to iron. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptor with tritiated spiperone to evaluate inhibition constants (K(i)). Functional activity of selected compounds was carried out with GTPgammaS binding assay. SAR results identified compounds (+)-19a and (-)-19b as two potent agonists for both D2 and D3 receptors (EC(50) (GTPgammaS); D2 = 4.51 and 1.69 nM and D3 = 1.58 and 0.74 nM for (-)-19b and (+)-19a, respectively). In vitro complexation studies with 19b demonstrated efficient chelation with iron. Furthermore, the deoxyribose assay with 19b demonstrated potent antioxidant activity. In PD animal model study, (-)-19b exhibited potent in vivo activity in reversing locomotor activity in reserpinized rats and also in producing potent rotational activity in 6-OHDA lesioned rats. This reports initial development of unique lead molecules that might find potential use in symptomatic and neuroprotective treatment of PD.
Assuntos
Quelantes de Ferro/farmacologia , Fármacos Neuroprotetores/farmacologia , Oxiquinolina/análogos & derivados , Oxiquinolina/farmacologia , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D3/agonistas , Animais , Modelos Animais de Doenças , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Quelantes de Ferro/síntese química , Quelantes de Ferro/química , Locomoção/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Masculino , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Oxiquinolina/síntese química , Doença de Parkinson/tratamento farmacológico , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/síntese química , Tetra-Hidronaftalenos/química , Tetra-Hidronaftalenos/farmacologiaRESUMO
The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Células Epiteliais/metabolismo , 1-Metil-4-fenilpiridínio/metabolismo , Ligação Competitiva/fisiologia , Linhagem Celular , Cocaína/análogos & derivados , Cocaína/metabolismo , Cocaína/farmacocinética , DNA Complementar/metabolismo , Dopamina/metabolismo , Dopamina/farmacocinética , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Norepinefrina/metabolismo , Ensaio Radioligante , Serotonina/metabolismoRESUMO
The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl(-)-dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na(+)-dependent conformational change, which may contribute to its Na(+)-dependent uptake inhibitory activity.
Assuntos
Ácido Aspártico/química , Cocaína/química , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/química , Proteínas do Tecido Nervoso , Alanina/química , Transporte Biológico , Biotinilação , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Cistina/química , Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Ácido Glutâmico/química , Humanos , Cinética , Ligantes , Mesilatos/farmacologia , Mutação , Fenótipo , Piperazinas/farmacologia , Ligação Proteica , Conformação Proteica , Transporte Proteico , Sódio/química , Fatores de Tempo , Zinco/química , Zinco/farmacologiaRESUMO
1. Conflicting results have been reported regarding the influence of nitric oxide (NO) and peroxynitrite on dopamine (DA) uptake and release. In the present study, effects of NO donors were studied in rat C6 glioma cells expressing human DA transporter. 2. [(3)H]-DA uptake was inhibited by S-nitroso-thiol S-nitroso-N-acetylpenicillamine, spermine/NO, diethylamine/NO (DEA/NO), (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO (PAPA/NO), and 3-morphosynodiomine (SIN-1) in a rank order correlating with their half lives as NO donors, whereas no effect was observed for diethylenetriamine/NO and dipropylenetriamine/NO, which release NO very slowly. 3. Hydroxycobalamin, a NO scavenger, but not superoxide dismutase and catalase, enzymes that metabolize superoxide and hydrogen peroxide, respectively, abolished the inhibitory effect of DEA/NO and SIN-1, indicating that they inhibit DA uptake through a mechanism related to the production of NO but unrelated to the formation of peroxynitrite. In consonance, peroxynitrite did not alter DA uptake in the present system. 4. DEA/NO and PAPA/NO reduced [(3)H]-MPP(+) uptake, whereas the release of [(3)H]-MPP(+) was not modified, demonstrating that NO can inhibit uptake of DA transporter substrate without accelerating DA transporter-mediated reverse transport of substrate under the same conditions.
Assuntos
1-Metil-4-fenilpiridínio/metabolismo , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/biossíntese , Proteínas do Tecido Nervoso , Óxido Nítrico/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina , Relação Dose-Resposta a Droga , Humanos , Doadores de Óxido Nítrico/farmacologia , Ratos , Células Tumorais CultivadasRESUMO
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) has been increasingly used as nitric oxide (NO) scavenger. Carboxy-PTIO reacts with NO to form nitric dioxide and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI). In rat C6 glioma cells expressing human dopamine transporter, carboxy-PTIO paradoxically potentiated the inhibition of [(3)H]dopamine uptake by two NO donors, diethylamine/NO and (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO. Further examinations revealed that carboxy-PTI concentration-dependently reduced dopamine uptake, indicating that the formation of carboxy-PTI may account for the failure of carboxy-PTIO to abolish NO elicited effects. These results suggest that caution should be taken in interpreting data obtained using carboxy-PTIO and probably other NO scavengers.