RESUMO
The dopamine transporter (DAT) plays an integral role in dopamine neurotransmission through the clearance of dopamine from the extracellular space. Dysregulation of DAT is central to the pathophysiology of numerous neuropsychiatric disorders and as such is an attractive therapeutic target. DAT belongs to the solute carrier family 6 (SLC6) class of Na+/Cl- dependent transporters that move various cargo into neurons against their concentration gradient. This review focuses on DAT (SCL6A3 protein) while extending the narrative to the closely related transporters for serotonin and norepinephrine where needed for comparison or functional relevance. Cloning and site-directed mutagenesis experiments provided early structural knowledge of DAT but our contemporary understanding was achieved through a combination of crystallization of the related bacterial transporter LeuT, homology modeling, and subsequently the crystallization of drosophila DAT. These seminal findings enabled a better understanding of the conformational states involved in the transport of substrate, subsequently aiding state-specific drug design. Post-translational modifications to DAT such as phosphorylation, palmitoylation, ubiquitination also influence the plasma membrane localization and kinetics. Substrates and drugs can interact with multiple sites within DAT including the primary S1 and S2 sites involved in dopamine binding and novel allosteric sites. Major research has centered around the question what determines the substrate and inhibitor selectivity of DAT in comparison to serotonin and norepinephrine transporters. DAT has been implicated in many neurological disorders and may play a role in the pathology of HIV and Parkinson's disease via direct physical interaction with HIV-1 Tat and α-synuclein proteins respectively.
RESUMO
BACKGROUND: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. OBJECTIVE: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. METHODS: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. RESULTS: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. CONCLUSIONS: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Distonia , Distúrbios Distônicos , Transtornos dos Movimentos , Transtornos do Neurodesenvolvimento , Animais , Distonia/diagnóstico , Distonia/genética , Distúrbios Distônicos/genética , Transtornos dos Movimentos/genética , Transtornos do Neurodesenvolvimento/genética , Prolina , RNA , Peixe-Zebra/genéticaRESUMO
The human dopamine transporter gene SLC6A3 has been consistently implicated in several neuropsychiatric diseases but the disease mechanism remains elusive. In this risk synthesis, we have concluded that SLC6A3 represents an increasingly recognized risk with a growing number of familial mutants associated with neuropsychiatric and neurological disorders. At least five loci were related to common and severe diseases including alcohol use disorder (high activity variant), attention-deficit/hyperactivity disorder (low activity variant), autism (familial proteins with mutated networking) and movement disorders (both regulatory variants and familial mutations). Association signals depended on genetic markers used as well as ethnicity examined. Strong haplotype selection and gene-wide epistases support multimarker assessment of functional variations and phenotype associations. Inclusion of its promoter region's functional markers such as DNPi (rs67175440) and 5'VNTR (rs70957367) may help delineate condensate-based risk action, testing a locus-pathway-phenotype hypothesis for one gene-multidisease etiology.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Proteínas da Membrana Plasmática de Transporte de Dopamina , Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Haplótipos , Humanos , Mutação , FenótipoRESUMO
We have carried out a structural exploration of (2S,4R,5R)-2-(bis(4-fluorophenyl)methyl)-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol (D-473) to investigate the influence of various functional groups on its aromatic ring, the introduction of heterocyclic aromatic rings, and the alteration of the stereochemistry of functional group on the pyran ring. The novel compounds were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting monoamine neurotransmitter uptake. Our studies identified some of the most potent dopamine-norepinephrine reuptake inhibitors known to-date like D-528 and D-529. The studies also led to development of potent triple reuptake inhibitors such as compounds D-544 and D-595. A significant influence from the alteration of the stereochemistry of the hydroxyl group on the pyran ring of D-473 on transporters affinities was observed indicating stereospecific preference for interaction. The inhibitory profiles and structure-activity relationship of lead compounds were further corroborated by molecular docking studies at the primary binding sites of monoamine transporters. The nature of interactions found computationally correlated well with their affinities for the transporters.
Assuntos
Dopamina , Norepinefrina , Proteínas da Membrana Plasmática de Transporte de Dopamina , Simulação de Acoplamento Molecular , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Piranos/farmacologia , Proteínas da Membrana Plasmática de Transporte de SerotoninaRESUMO
Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.
Assuntos
Proteínas de Transporte de Neurotransmissores/química , Proteínas de Transporte de Neurotransmissores/metabolismo , Animais , HumanosRESUMO
There is a plethora of amphetamine derivatives exerting stimulant, euphoric, anti-fatigue, and hallucinogenic effects; all structural properties allowing these effects are contained within the amphetamine structure. In the first part of this review, the interaction of amphetamine with the dopamine transporter (DAT), crucially involved in its behavioral effects, is covered, as well as the role of dopamine synthesis, the vesicular monoamine transporter VMAT2, and organic cation 3 transporter (OCT3). The second part deals with requirements in amphetamine's effect on the kinases PKC, CaMKII, and ERK, whereas the third part focuses on where we are in developing anti-amphetamine therapeutics. Thus, treatments are discussed that target DAT, VMAT2, PKC, CaMKII, and OCT3. As is generally true for the development of therapeutics for substance use disorder, there are multiple preclinically promising specific compounds against (meth)amphetamine, for which further development and clinical trials are badly needed.
Assuntos
Anfetamina/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/fisiologia , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Proteínas Vesiculares de Transporte de Monoamina/fisiologia , HumanosRESUMO
Significant unmet needs exist for development of better pharmacotherapeutic agents for major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) as the current drugs are inadequate. Our goal in this study is to investigate behavioral pharmacological characterization of a novel triple reuptake inhibitor (TRI) D-578 which exhibits nanomolar potency at all three monoamine transporters (Ki; 16.2. 16.2, 3.23â¯nM, and 29.6, 20.6, 6.10â¯nM for the rat brain and cloned human dopamine, serotonin and norepinephrine transporters, respectively) and exhibited little to no affinity for other off-target CNS receptors. In a rat forced swim test, compound D-578 upon oral administration displayed high efficacy and not stimulating in locomotor behavior. The effects of D-578 and paroxetine were next evaluated in a rat model for traumatic stress exposure - the single prolonged stress (SPS) model - which has been shown to have construct, predictive, and behavioral validity in modeling aspects of PTSD. Our results show that SPS had no effect on the acquisition of conditioned fear, but impaired extinction learning and extinction retention of fear behavior compared to sham treatment. D-578, but not paroxetine, attenuated the extinction and extinction-retention deficit induced by SPS. These findings suggest that D-578 has greater efficacy in normalizing traumatic stress-induced extinction-retention learning in a model for PTSD compared to paroxetine. Overall these results suggest that D-578, in addition to producing a robust and efficacious antidepressant effect, may attenuate maladaptive retention of fearful memories and support further testing of this agent for the pharmacotherapy of depression and PTSD.
Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo Maior/tratamento farmacológico , Inibidores da Captação de Neurotransmissores/farmacologia , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológico , Estresse Psicológico/complicações , Administração Oral , Animais , Antidepressivos/uso terapêutico , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Transtorno Depressivo Maior/etiologia , Transtorno Depressivo Maior/psicologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Inibidores da Captação de Neurotransmissores/uso terapêutico , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Ratos , Retenção Psicológica/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Transtornos de Estresse Pós-Traumáticos/etiologia , Transtornos de Estresse Pós-Traumáticos/psicologia , Estresse Psicológico/psicologiaRESUMO
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
Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 - a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of â¼500â¯nM and behaves as a NET substrate. Systemic dosing of SK609 (4â¯mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4â¯mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05â¯mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25â¯mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8â¯mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.
Assuntos
Atenção/fisiologia , Butilaminas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Receptores de Dopamina D3/fisiologia , Animais , Butilaminas/antagonistas & inibidores , Células Cultivadas , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Masculino , Atividade Motora/efeitos dos fármacos , Norepinefrina/metabolismo , Prazosina/farmacologia , Córtex Pré-Frontal/metabolismo , Racloprida/farmacologia , Ratos , Receptores de Dopamina D3/agonistasRESUMO
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
A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [3H]DA and inhibitor [3H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [3H]DA and [3H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.
Assuntos
Deficiências do Desenvolvimento/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Sítios de Ligação , Cristalografia por Raios X , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Humanos , Modelos Moleculares , MutaçãoRESUMO
Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross-linked natural DAT dimers in LLC-PK1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross-linked by CuP showed normal DA uptake with DA Km at ~ 0.5 µM and DA efflux with basal and amphetamine-induced DA efflux as much as control values. The cocaine analog 2ß-carbomethoxy-3ß-[4-fluorophenyl]-tropane (CFT) was capable to bind to copper-cross-linked DATs, albeit with an affinity more than fivefold decreased (Kd of CFT = 109 nM after cross-linking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Fenantrolinas/química , Animais , Biotinilação , Cocaína/química , Cocaína/metabolismo , Reagentes de Ligações Cruzadas/química , Inibidores da Captação de Dopamina/química , Inibidores da Captação de Dopamina/metabolismo , Células HEK293 , Humanos , Cinética , Células LLC-PK1 , Ligação Proteica , Suínos , TropanosRESUMO
The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.
Assuntos
Anfetamina/farmacologia , Transporte Biológico/efeitos dos fármacos , Cocaína/metabolismo , Dopaminérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/metabolismo , Dopamina/metabolismo , Animais , Linhagem Celular , Cocaína/análogos & derivados , Inibidores da Captação de Dopamina/química , Masculino , Metanfetamina/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Suínos , Treonina/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
Dopamine receptors play an integral role in controlling brain physiology. Importantly, subtype selective agonists and antagonists of dopamine receptors with biased signaling properties have been successful in treating psychiatric disorders with a low incidence of side effects. To this end, we recently designed and developed SK609, a dopamine D3 receptor (D3R) selective agonist that has atypical signaling properties. SK609 has shown efficacy in reversing akinesia and reducing L-dopa-induced dyskinesia in a hemiparkinsonian rats. In the current study, we demonstrate that SK609 has high selectivity for D3R with no binding affinity on D2R high- or low-affinity state when tested at a concentration of 10 µM. In addition, SK609 and its analogues do not induce desensitization of D3R as determined by repeated agonist treatment response in phosphorylation of ERK1/2 functional assay. Most significantly, SK609 and its analogues preferentially signal through the G-protein-dependent pathway and do not recruit ß-arrestin-2, suggesting a functional bias toward the G-protein-dependent pathway. Structure-activity relationship (SAR) studies using analogues of SK609 demonstrate that the molecules bind at the orthosteric site by maintaining the conserved salt bridge interactions with aspartate 110 on transmembrane 3 and aryl interactions with histidine 349 on transmembrane 6, in addition to several hydrophobic interactions with residues from transmembranes 5 and 6. The compounds follow a strict SAR with reference to the three pharmacophore elements: substituted phenyl ring, length of the linker connecting phenyl ring and amine group, and orientation and hydrophobic branching groups at the amine among SK609 analogues for efficacy and functional selectivity. These features of SK609 and the analogues suggest that biased signaling is an inherent property of this series of molecules.
Assuntos
Butilaminas/química , Butilaminas/farmacologia , Receptores de Dopamina D3/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Benzazepinas/farmacocinética , Células CHO , Cricetulus , Antagonistas de Dopamina/farmacocinética , Antagonistas de Dopamina/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Células HEK293 , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Transdução de Sinais/genética , Isótopos de Enxofre/farmacocinética , Transfecção , Trítio/farmacocinéticaRESUMO
Food restriction (FR) and obesogenic (OB) diets are known to alter brain dopamine transmission and exert opposite modulatory effects on behavioral responsiveness to psychostimulant drugs of abuse. Mechanisms underlying these diet effects are not fully understood. In this study, we examined diet effects on expression and function of the dopamine transporter (DAT) in caudate-putamen (CPu), nucleus accumbens (NAc), and midbrain regions. Dopamine (DA) uptake by CPu, NAc or midbrain synapto(neuro)somes was measured in vitro with rotating disk electrode voltammetry or with [3 H]DA uptake and was found to correlate with DAT surface expression, assessed by maximal [3 H](-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane binding and surface biotinylation assays. FR and OB diets were both found to decrease DAT activity in CPu with a corresponding decrease in surface expression but had no effects in the NAc and midbrain. Diet treatments also affected sensitivity to insulin-induced enhancement of DA uptake, with FR producing an increase in CPu and NAc, likely mediated by an observed increase in insulin receptor expression, and OB producing a decrease in NAc. The increased expression of insulin receptor in NAc of FR rats was accompanied by increased DA D2 receptor expression, and the decreased DAT expression and function in CPu of OB rats was accompanied by decreased DA D2 receptor expression. These results are discussed as partial mechanistic underpinnings of diet-induced adaptations that contribute to altered behavioral sensitivity to psychostimulants that target the DAT.
Assuntos
Química Encefálica/efeitos dos fármacos , Dieta , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Animais , Biotinilação , Peso Corporal , Restrição Calórica , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Obesidade/metabolismo , Putamen/efeitos dos fármacos , Putamen/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
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.