RESUMO
Due to its inherent reactivity, HNO must be generated in situ through the use of donor compounds. One of the primary strategies for the development of new HNO donors has been modifying hydroxylamines with good leaving groups. A recent example of this strategy is the (hydroxylamino)barbituric acid (HABA) class of HNO donors. In this case, however, an undesired intramolecular rearrangement pathway to the corresponding hydantoin derivative competes with HNO formation, particularly in the absence of chemical traps for HNO. This competitive non-HNO-producing pathway has restricted the development of the HABA class to examples with fast HNO release profiles at physiological pH and temperature (t(1/2) < 1 min). Herein, the factors that favor the rearrangement pathway have been examined and two independent strategies that protect against rearrangement to favor HNO generation have been developed. The timecourse and stoichiometry for the in vitro conversion of these compounds to HNO (trapped as a phosphine aza-ylide) and the corresponding barbituric acid (BA) byproduct have been determined by (1)H NMR spectroscopy under physiologically relevant conditions. These results confirm the successful extension of the HABA class of pure HNO donors with half-lives at pH 7.4, 37 °C ranging from 19 to 107 min.
Assuntos
Barbitúricos/química , Óxidos de Nitrogênio/química , Fenômenos Bioquímicos , Hidroxilaminas/química , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
A new and versatile class of HNO donors, the (hydroxylamino)pyrazolone (HAPY) series of HNO donors utilizing pyrazolone (PY) leaving groups, is described. HNO, the smallest N-based aldehyde equivalent, is used as a reagent along with a variety of PY compounds to synthesize the desired HAPY donors in what can be considered an N-selective HNO-aldol reaction in up to quantitative yields. The bimolecular rate constant of HNO with PY in pH 7.4 phosphate buffer at 37 °C can reach 8 × 10(5) M(-1) s(-1). In (1)H NMR experiments, the HAPY compounds generate HNO quantitatively (trapped as a phosphine aza-ylide) with half-lives spanning 3 orders of magnitude (minutes to days) under physiologically relevant conditions. B3LYP/6-31G* calculations confirm the energetically favorable reactions between HNO and the PY enol and enolate, whereas HNO release is expected to occur through the oxyanion (OHN-PY) of each HAPY compound. HNO has been shown to provide functional support to failing hearts.
Assuntos
Óxidos de Nitrogênio/química , Pirazolonas/química , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Due to its inherent reactivity, nitroxyl (HNO), must be generated in situ through the use of donor compounds, but very few physiologically useful HNO donors exist. Novel N-substituted hydroxylamines with carbon-based leaving groups have been synthesized, and their structures confirmed by X-ray crystallography. These compounds generate HNO under nonenzymatic, physiological conditions, with the rate and amount of HNO released being dependent mainly on the nature of the leaving group. A barbituric acid and a pyrazolone derivative have been developed as efficient HNO donors with half-lives at pH 7.4, 37 °C of 0.7 and 9.5 min, respectively.
Assuntos
Hidroxilaminas/química , Óxidos de Nitrogênio/síntese química , Cristalografia por Raios X , Hidroxilaminas/síntese química , Modelos Moleculares , Estrutura Molecular , Óxidos de Nitrogênio/químicaRESUMO
The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca2+ influx via N-type voltage-gated Ca2+ channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.
Assuntos
Autorreceptores , Proteínas da Membrana Plasmática de Transporte de Dopamina , Animais , Autorreceptores/metabolismo , Clatrina/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Camundongos , Fosfatidilinositóis/metabolismo , Proteínas Qa-SNARE/metabolismoRESUMO
Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG601,602AA and DAT-PG584,585AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo in Drosophila harboring the DAT-PG584,585AA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.
RESUMO
The utilization of fluorescent ligands to study the monoamine transporters (MATs) has increased our knowledge of their function and distribution in live cell systems. In this study, we extend SAR for nisoxetine and talopram as parent compounds, to identify high affinity rhodamine-labeled fluorescent probes for the norepinephrine transporter (NET). Nisoxetine-based fluorescent probe 6 demonstrated high binding affinity (K i = 43 nM) for NET and an overall selectivity compared to the other transporters for dopamine (DAT; K i = 1540 nM) and serotonin (SERT; K i = 785 nM) in competitive radioligand binding assays. Using confocal microscopy, compound 6 was shown to stain both NET and SERT, but not DAT, at low nanomolar concentrations, in transporter-expressing cells.
RESUMO
The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and is the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.
Assuntos
Cocaína , Proteínas da Membrana Plasmática de Transporte de Dopamina , Animais , Dopamina , Inibidores da Captação de Dopamina , Ligantes , Ratos , Imagem Individual de MoléculaRESUMO
Conformational influences profoundly impact the performance of organic electronic materials and the reactivity of organic molecules. We recently found that the expected former consideration was uniquely accompanied by the latter. This report describes a surprisingly regioselective bromination that suggests the general use of conformation as a protecting group during complex molecule synthesis.
Assuntos
Conformação Molecular , Polímeros/química , Polímeros/síntese química , Elétrons , Fenômenos de Química Orgânica , Fotoquímica , EstereoisomerismoRESUMO
In contrast to temporal coding by synaptically acting neurotransmitters such as glutamate, neuromodulators such as monoamines signal changes in firing rate. The two modes of signaling have been thought to reflect differences in release by different cells. We now find that midbrain dopamine neurons release glutamate and dopamine with different properties that reflect storage in different synaptic vesicles. The vesicles differ in release probability, coupling to presynaptic Ca2+ channels and frequency dependence. Although previous work has attributed variation in these properties to differences in location or cytoskeletal association of synaptic vesicles, the release of different transmitters shows that intrinsic differences in vesicle identity drive different modes of release. Indeed, dopamine but not glutamate vesicles depend on the adaptor protein AP-3, revealing an unrecognized linkage between the pathway of synaptic vesicle recycling and the properties of exocytosis. Storage of the two transmitters in different vesicles enables the transmission of distinct signals.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Canais de Cálcio/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Exocitose , Ácido Glutâmico/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Mesencéfalo/citologia , Camundongos , Neurônios/metabolismo , Neurotransmissores/metabolismoRESUMO
The sigma 1 receptor (σ1R) is a structurally unique transmembrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER), and has been implicated in cancer, neuropathic pain, and psychostimulant abuse. Despite physiological and pharmacological significance, mechanistic underpinnings of structure-function relationships of σ1R are poorly understood, and molecular interactions of selective ligands with σ1R have not been elucidated. The recent crystallographic determination of σ1R as a homo-trimer provides the foundation for mechanistic elucidation at the molecular level. Here we report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of σ1R and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σ1R homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σ1R multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σ1R multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σ1R-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σ1R in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σ1R upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze σ1R-ligand binding and its impact on interaction of σ1R with client proteins.
Assuntos
Ligantes , Receptores sigma/química , Receptores sigma/metabolismo , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/metabolismo , Antagonistas de Dopamina/farmacologia , Cobaias , Células HEK293 , Haloperidol/análogos & derivados , Haloperidol/farmacocinética , Haloperidol/farmacologia , Humanos , Isoxazóis/farmacologia , Masculino , Simulação de Acoplamento Molecular , Pentazocina/farmacocinética , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Piridinas/farmacologia , Receptores sigma/genética , Transfecção , Trítio/farmacocinética , Receptor Sigma-1RESUMO
Dopamine transporter (DAT) has been shown to accumulate in filopodia in neurons and non-neuronal cells. To examine the mechanisms of DAT filopodial targeting, we used quantitative live-cell fluorescence microscopy, and compared the effects of the DAT inhibitor cocaine and its fluorescent analog JHC1-64 on the plasma membrane distribution of wild-type DAT and two non-functional DAT mutants, R60A and W63A, that do not accumulate in filopodia. W63A did not bind JHC1-64, whereas R60A did, although less efficiently compared to the wild-type DAT. Molecular dynamics simulations predicted that R60A preferentially assumes an outward-facing (OF) conformation through compensatory intracellular salt bridge formation, which in turn favors binding of cocaine. Imaging analysis showed that JHC1-64-bound R60A mutant predominantly localized in filopodia, whereas free R60A molecules were evenly distributed within the plasma membrane. Cocaine binding significantly increased the density of R60A, but not that of W63A, in filopodia. Further, zinc binding, known to stabilize the OF state, also increased R60A concentration in filopodia. Finally, amphetamine, that is thought to disrupt DAT OF conformation, reduced the concentration of wild-type DAT in filopodia. Altogether, these data indicate that OF conformation is required for the efficient targeting of DAT to, and accumulation in, filopodia.
Assuntos
Anfetamina/farmacologia , Cloretos/farmacologia , Cocaína/farmacologia , Dextroanfetamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Compostos de Zinco/farmacologia , Anfetamina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cloretos/metabolismo , Cocaína/análogos & derivados , Cocaína/metabolismo , Dextroanfetamina/metabolismo , Dopamina/metabolismo , Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação Proteica , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Compostos de Zinco/metabolismoRESUMO
The development of medications to treat cocaine use disorders has thus far defied success, leaving this patient population without pharmacotherapeutic options. As the dopamine transporter (DAT) plays a prominent role in the reinforcing effects of cocaine that can lead to addiction, atypical DAT inhibitors have been developed that prevent cocaine from binding to DAT, but they themselves are not cocaine-like. Herein, a series of novel DAT inhibitors were synthesized, and based on its pharmacological profile, the lead compound 10a was evaluated in phase I metabolic stability studies in mouse liver microsomes and compared to cocaine in locomotor activity and drug discrimination paradigms in mice. A molecular dynamic simulation study supported the hypothesis that atypical DAT inhibitors have similar binding poses at DAT in a conformation that differs from that of cocaine. Such differences may ultimately contribute to their unique behavioral profiles and potential for development as cocaine use disorder therapeutics.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Relação Estrutura-Atividade , Animais , Benzotropina/química , Células COS , Chlorocebus aethiops , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Simulação de Dinâmica Molecular , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos Sprague-Dawley , Tropanos/químicaRESUMO
A new strategy to achieve regioselective functionalization of a sterically congested aromatic system driven by conformational demands is described. Electrophilic substitution occurs at the more planarizable subunit without undesired chemistry at mutually reactive sites and without the need for protecting or masking groups that must be manipulated later. Model studies are described to understand this selectivity, and possibilities for the construction of orthogonal, differentially substituted pi-systems of relevance for molecular electronics are demonstrated.