RESUMO
The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson's disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2's importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here, we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington's chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.
Assuntos
Doença de Huntington , Tetrabenazina , Humanos , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Prótons , Microscopia CrioeletrônicaRESUMO
Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.
Assuntos
Tetrabenazina , Proteínas Vesiculares de Transporte de Monoamina , Humanos , Reserpina , Serotonina/metabolismo , Vesículas Sinápticas/metabolismo , Tetrabenazina/farmacologia , Tetrabenazina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission1-3. Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression4-6. Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease7,8, respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents.
Assuntos
Microscopia Crioeletrônica , Proteínas Vesiculares de Transporte de Monoamina , Humanos , Sítios de Ligação , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ketanserina/química , Ketanserina/metabolismo , Ketanserina/farmacologia , Reserpina/química , Reserpina/metabolismo , Reserpina/farmacologia , Serotonina/química , Serotonina/metabolismo , Especificidade por Substrato , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/ultraestruturaRESUMO
Noninvasive quantitative imaging of beta-cells can provide information on changes in cellular transporters, receptors, and signaling proteins that may affect function and/or loss of mass, both of which contribute to the loss of insulin secretion and glucose regulation of patients with type 1 or type 2 diabetes (T1D/T2D). We have developed and optimized the use of two positron emission tomography (PET) radioligands, [18F]FP-(+)-DTBZ and [11C](+)-PHNO, targeting beta-cell VMAT2 and dopamine (D2/D3) receptors, respectively. Here we describe our optimized methodology for the clinical use of these two tracers for quantitative PET imaging of beta-cell biomarkers in vivo. We also briefly discuss our previous results and their implications and value towards extending the use of PET radioligand beyond the original goal of quantitative imaging of beta-cell mass to the potential to provide insight into the biology of beta-cell loss of mass and/or function and to evaluate the efficacy of therapeutics to prevent or restore functional beta-cell mass.
Assuntos
Diabetes Mellitus Tipo 2 , Receptores de Dopamina D3 , Humanos , Receptores de Dopamina D3/metabolismo , Dopamina , Receptores de Dopamina D2/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico por imagem , Diabetes Mellitus Tipo 2/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Tetrabenazina/metabolismoRESUMO
BACKGROUND: L-DOPA-induced dyskinesia (LID), occurring with aberrant processing of exogenous L-DOPA in the dopamine-denervated striatum, is a main complication of levodopa treatment in Parkinson's disease. OBJECTIVE: To characterize the effects of the vesicular antagonist tetrabenazine (TBZ) on L-DOPA-induced behavior, neurochemical signals, and underlying protein expressions in an animal model of Parkinson's disease. METHODS: 20-week-old MitoPark mice were co-treated or separately administered TBZ and L-DOPA for 14 days. Abnormal involuntary movements (AIMs) and locomotor activity were analyzed. To explore dopamine (DA) transmission, fast scan cyclic voltammetry was used to assess presynaptic DA dynamics in striatal slices following treatments. PET imaging with 4-[18F]-PE2I, ADAM and immunoblotting assays were used to detect receptor protein changes in the DA-denervated striatum. Finally, nigrostriatal tissues were collected for HPLC measures of DA, serotonin and their metabolites. RESULTS: A single injection of TBZ given in the interval between the two L-DOPA/Carbidopa treatments significantly attenuated L-DOPA-induced AIMs expression and locomotor hyperactivity. TBZ was shown to reduce tonic and phasic release of DA following L-DOPA treatment in DA-denervated striatal tissue. In the DA-depleted striatum, TBZ decreased the expression of L-DOPA-enhanced D1 receptors and the serotonin reuptake transporter. Neurochemical analysis indicated that TBZ attenuated L-DOPA-induced surges of DA levels by promoting DA turnover in the nigrostriatal system. CONCLUSIONS: Our findings demonstrate that TBZ diminishes abnormal striatal DA transmission, which involves the ability of TBZ to modulate the presymptomatic dynamics of DA, and then mitigate aberrant release of exogenous L-DOPA from nerve terminals. The results support the potential of repositioning TBZ to counteract LID development.
Assuntos
Discinesia Induzida por Medicamentos , Doença de Parkinson , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Discinesia Induzida por Medicamentos/etiologia , Levodopa/efeitos adversos , Camundongos , Oxidopamina/metabolismo , Oxidopamina/farmacologia , Doença de Parkinson/complicações , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Serotonina/farmacologia , Tetrabenazina/metabolismo , Tetrabenazina/farmacologiaRESUMO
Vesicular monoamine transporter 2 (VMAT2) is essential for synaptic transmission of all biogenic amines in the brain including serotonin, norepinephrine, histamine, and dopamine (DA). Given its crucial role in the neurophysiology and pharmacology of the central nervous system, VMAT2 is recognized as an important therapeutic target for various neurological disorders such as tardive dyskinesia (TD). Here, a novel series of dihydrotetrabenazine derivative analogs were designed and synthesized to evaluate their effects on [3H]dihydrotetrabenazine (DTBZ) binding and [3H]DA uptake at VMAT2. Of these analogs, compound 13e showed a high binding affinity for VMAT2 (IC50 = 5.13 ± 0.16 nM) with excellent inhibition of [3H]DA uptake (IC50 = 6.04 ± 0.03 nM) in striatal synaptosomes. In human liver microsomes, 13e was more stable (T1/2 = 161.2 min) than other reported VMAT2 inhibitors such as DTBZ (T1/2 = 119.5 min). In addition, 13e effectively inhibited the spontaneous locomotor activity (percent inhibition at 3 µmol/kg = 64.7%) in Sprague-Dawley rats. Taken together, our results indicate that 13e might be a promising lead compound for the development of novel treatments of TD.
Assuntos
Tetrabenazina/análogos & derivados , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Animais , Humanos , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Tetrabenazina/uso terapêuticoRESUMO
INTRODUCTION: In vivo imaging methods such as Positron Emission Tomography (PET) can be used to examine the relationship between in vitro binding affinity and in vivo occupancy of binding sites in the brain for new drug candidates. In this study, PET imaging in monkey brain was used to evaluate that correlation for a set of four diastereomers of the compound dihydrotetrabenazine (DTBZ), the pharmacologically active metabolite of the drug tetrabenazine. METHODS: PET studies of DTBZ diastereomers were completed in a single monkey brain. In vivo occupancies (ED50) were estimated using multiple drug doses and the vesicular monoamine transporter 2 specific radioligand (+)-α-[11C] DTBZ, employing a test-retest sequence of control PET scan, drug administration and a second PET scan completed on a single day. RESULTS: DTBZ has three chiral carbon centers and eight possible stereoisomers, and in vivo occupancy of the target site VMAT2 was observed only for the four diastereomers of DTBZ having the 11bR absolute configuration. The estimated in vivo occupancies (ED50 values from 0.023 to >3.15 mg/kg) correlated well (R2 = 0.95) with the in vitro binding affinities (Ki values of 4 to 600 nM for the VMAT2), and an even better correlation (R2 = 0.99) was found for the three isomers with in vitro binding affinities <100 nM. CONCLUSIONS: If the physiochemical (MW, log P, pKa) or physiological (metabolism, transport, protein binding) properties of a set of drug stereoisomers are considered similar, the binding affinities determined from in vitro assays may predict the in vivo occupancies of the target binding site in the monkey brain.
Assuntos
Tomografia por Emissão de Pósitrons/métodos , Tetrabenazina/análogos & derivados , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Radioisótopos de Flúor , Haplorrinos , Masculino , Ligação Proteica , Estereoisomerismo , Tetrabenazina/química , Tetrabenazina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
RATIONALE: Atypical dopamine (DA) transport blockers such as modafinil and its analogs may be useful for treating motivational symptoms of depression and other disorders. Previous research has shown that the DA depleting agent tetrabenazine can reliably induce motivational deficits in rats, as evidenced by a shift towards a low-effort bias in effort-based choice tasks. This is consistent with human studies showing that people with major depression show a bias towards low-effort activities. OBJECTIVES: Recent studies demonstrated that the atypical DA transport (DAT) inhibitor (S)-CE-123 reversed tetrabenazine-induced motivational deficits, increased progressive ratio (PROG) lever pressing, and increased extracellular DA in the nucleus accumbens. In the present studies, a recently synthesized modafinil analog, (S, S)-CE-158, was assessed in a series of neurochemical and behavioral studies in rats. RESULTS: (S, S)-CE-158 demonstrated the ability to reverse the effort-related effects of tetrabenazine and increase selection of high-effort PROG lever pressing in rats tested on PROG/chow feeding choice task. (S, S)-CE-158 showed a high selectivity for inhibiting DAT compared with other monoamine transporters, and systemic administration of (S, S)-CE-158 increased extracellular DA in the nucleus accumbens during the behaviorally active time course, which is consistent with the effects of (S)-CE-123 and other DAT inhibitors that enhance high-effort responding. CONCLUSIONS: These studies provide an initial neurochemical characterization of a novel atypical DAT inhibitor, and demonstrate that this compound is active in models of effort-related choice. This research could contribute to the development of novel compounds for the treatment of motivational dysfunctions in humans.
Assuntos
Comportamento de Escolha/fisiologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Comportamento Alimentar/fisiologia , Modafinila/análogos & derivados , Modafinila/metabolismo , Motivação/fisiologia , Inibidores da Captação Adrenérgica/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Animais , Comportamento de Escolha/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Comportamento Alimentar/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Modafinila/farmacologia , Motivação/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Tetrabenazina/metabolismo , Tetrabenazina/farmacologiaRESUMO
OBJECTIVE: To delineate the natural history, diagnosis, and treatment response of Parkinson disease (PD) in individuals with 22q11.2 deletion syndrome (22q11.2DS), and to determine if these patients differ from those with idiopathic PD. METHODS: In this international observational study, we characterized the clinical and neuroimaging features of 45 individuals with 22q11.2DS and PD (mean follow-up 7.5 ± 4.1 years). RESULTS: 22q11.2DS PD had a typical male excess (32 male, 71.1%), presentation and progression of hallmark motor symptoms, reduced striatal dopamine transporter binding with molecular imaging, and initial positive response to levodopa (93.3%). Mean age at motor symptom onset was relatively young (39.5 ± 8.5 years); 71.4% of cases had early-onset PD (<45 years). Despite having a similar age at onset, the diagnosis of PD was delayed in patients with a history of antipsychotic treatment compared with antipsychotic-naive patients (median 5 vs 1 year, p = 0.001). Preexisting psychotic disorders (24.5%) and mood or anxiety disorders (31.1%) were common, as were early dystonia (19.4%) and a history of seizures (33.3%). CONCLUSIONS: Major clinical characteristics and response to standard treatments appear comparable in 22q11.2DS-associated PD to those in idiopathic PD, although the average age at onset is earlier. Importantly, treatment of preexisting psychotic illness may delay diagnosis of PD in 22q11.DS patients. An index of suspicion and vigilance for complex comorbidity may assist in identifying patients to prioritize for genetic testing.
Assuntos
Síndrome de DiGeorge/complicações , Síndrome de DiGeorge/diagnóstico , Doença de Parkinson/complicações , Doença de Parkinson/diagnóstico , Adulto , Antiparkinsonianos/uso terapêutico , Bases de Dados Bibliográficas/estatística & dados numéricos , Estimulação Encefálica Profunda , Síndrome de DiGeorge/mortalidade , Síndrome de DiGeorge/terapia , Feminino , Humanos , Cooperação Internacional , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/mortalidade , Doença de Parkinson/terapia , Índice de Gravidade de Doença , Distribuição por Sexo , Estatísticas não Paramétricas , Tetrabenazina/análogos & derivados , Tetrabenazina/metabolismo , Tomografia Computadorizada de Emissão de Fóton ÚnicoAssuntos
Inibidores da Captação Adrenérgica/química , Deutério/química , Aprovação de Drogas , Descoberta de Drogas , Tetrabenazina/análogos & derivados , Inibidores da Captação Adrenérgica/efeitos adversos , Inibidores da Captação Adrenérgica/metabolismo , Inibidores da Captação Adrenérgica/uso terapêutico , Animais , Bioquímica/métodos , Coreia/tratamento farmacológico , Deutério/efeitos adversos , Deutério/metabolismo , Deutério/uso terapêutico , Aprovação de Drogas/métodos , Descoberta de Drogas/métodos , Controle de Medicamentos e Entorpecentes/métodos , Humanos , Discinesia Tardia/tratamento farmacológico , Tetrabenazina/efeitos adversos , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/uso terapêuticoRESUMO
INTRODUCTION: The longitudinal reproducibility of in vivo binding potential measures for [11C]dihydrotetrabenazine ([11C]DTBZ) binding to the vesicular monoamine transporter 2 (VMAT2) site in primate brain was examined using a unique dataset of repeated control PET imaging studies. METHODS: Forty-one dynamic [11C]DTBZ PET studies were completed in a single rhesus monkey. Imaging equipment (microPET P4), personnel, radiotracer characteristics (injected mass amounts, molar activity) and image data analysis (BPND-Logan) were consistent throughout the entire sequence of PET studies. RESULTS: Same day reproducibility of BPND-Logan estimates of specific binding was very good (-3% and -7% changes) for two control-control sessions. Over the full 74 months, the average BPND-Logan value for [11C]DTBZ-PET studies was 4.19±0.52, for a variance of 12%. No age-dependent change in binding potentials was observed over the six-year period. CONCLUSIONS: If the technical variables associated with PET scanner are consistently maintained, including PET scanner, imaging procedures and radiotracer preparation, in vivo biochemistry can be reproducibly measured in the primate brain over a multi-year period of time.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Tomografia por Emissão de Pósitrons , Tetrabenazina/análogos & derivados , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Estudos Longitudinais , Macaca mulatta , Ligação Proteica , Radioquímica , Tetrabenazina/química , Tetrabenazina/metabolismoRESUMO
The recurrent 22q11.2 deletion is a genetic risk factor for early-onset Parkinson's disease. Adults with the associated 22q11.2 deletion syndrome (22q11.2DS) may exhibit phenotypes that could help identify those at highest risk and reveal disease trajectories. We investigated clinical and neuroimaging features relevant to Parkinson's disease in 26 adults: 13 with 22q11.2DS at genetic risk of Parkinson's disease (mean age = 41.5 years, standard deviation = 9.7), 12 healthy age and sex-matched controls, and a 22q11.2DS patient with l-DOPA-responsive early-onset Parkinson's disease. Neuroimaging included transcranial sonography and positron emission tomography using 11C-dihydrotetrabenazine (11C-DTBZ), a radioligand that binds to the presynaptic vesicular monoamine transporter. The 22q11.2DS group without Parkinson's disease demonstrated significant motor and olfactory deficits relative to controls. Eight (61.5%) were clinically classified with parkinsonism. Transcranial sonography showed a significantly larger mean area of substantia nigra echogenicity in the 22q11.2DS risk group compared with controls (P = 0.03). The 22q11.2DS patient with Parkinson's disease showed the expected pattern of severely reduced striatal 11C-DTBZ binding. The 22q11.2DS group without Parkinson's disease however showed significantly elevated striatal 11C-DTBZ binding relative to controls (â¼33%; P < 0.01). Results were similar within the 22q11.2DS group for those with (n = 7) and without (n = 6) psychotic illness. These findings suggest that manifestations of parkinsonism and/or evolution to Parkinson's disease in this genetic at-risk population may include a hyperdopaminergic mechanism. Adequately powered longitudinal studies and animal models are needed to evaluate the relevance of the observed clinical and imaging phenotypes to Parkinson's disease and other disorders that are more prevalent in 22q11.2DS, such as schizophrenia.
Assuntos
Síndrome de DiGeorge/complicações , Síndrome de DiGeorge/fisiopatologia , Neuroimagem Funcional , Doença de Parkinson Secundária/diagnóstico , Doença de Parkinson Secundária/fisiopatologia , Adulto , Radioisótopos de Carbono/metabolismo , Estudos de Casos e Controles , Corpo Estriado/metabolismo , Síndrome de DiGeorge/genética , Feminino , Humanos , Hipertrofia/patologia , Masculino , Doença de Parkinson Secundária/complicações , Doença de Parkinson Secundária/genética , Tomografia por Emissão de Pósitrons , Substância Negra/patologia , Tetrabenazina/análogos & derivados , Tetrabenazina/metabolismo , Ultrassonografia Doppler TranscranianaRESUMO
The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2, is approved for the treatment of tardive dyskinesia. Valbenazine is converted to two significant circulating metabolites in vivo, namely, (+)-α-dihydrotetrabenazine (R,R,R-HTBZ) and a mono-oxy metabolite, NBI-136110. Radioligand-binding studies were conducted to assess and compare valbenazine, tetrabenazine, and their respective metabolites in their abilities to selectively and potently inhibit [3H]-HTBZ binding to VMAT2 in rat striatal, rat forebrain, and human platelet homogenates. A broad panel screen was conducted to evaluate possible off-target interactions of valbenazine, R,R,R-HTBZ, and NBI-136110 at >80 receptor, transporter, and ion channel sites. Radioligand binding showed R,R,R-HTBZ to be a potent VMAT2 inhibitor in homogenates of rat striatum (Ki = 1.0-2.8 nM), rat forebrain (Ki = 4.2 nM), and human platelets (Ki = 2.6-3.3 nM). Valbenazine (Ki = 110-190 nM) and NBI-136110 (Ki = 160-220 nM) also exhibited inhibitory effects on VMAT2, but with lower potency than R,R,R-HTBZ. Neither valbenazine, R,R,R-HTBZ, nor NBI-136110 had significant off-target interactions at serotonin (5-HT1A, 5-HT2A, 5-HT2B) or dopamine (D1 or D2) receptor sites. In vivo studies measuring ptosis and prolactin secretion in the rat confirmed the specific and dose-dependent interactions of tetrabenazine and R,R,R-HTBZ with VMAT2. Evaluations of potency and selectivity of tetrabenazine and its pharmacologically active metabolites were also performed. Overall, the pharmacologic characteristics of valbenazine appear consistent with the favorable efficacy and tolerability findings of recent clinical studies [KINECT 2 (NCT01733121), KINECT 3 (NCT02274558)].
Assuntos
Tetrabenazina/análogos & derivados , Valina/análogos & derivados , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células CHO , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Valina/metabolismo , Valina/farmacologiaRESUMO
OBJECTIVE: Lactacystin has been used to establish rodent models of Parkinson disease (PD), with cerebral α-synuclein inclusions. This study evaluated the uptake of [18F]9-fluoropropyl-(+)-dihydrotetrabenazine ([18F]FP-(+)-DTBZ), a vesicular monoamine transporter type 2 (VMAT2)-targeting radiotracer, through positron emission tomography (PET) in lactacystin-treated rat brains. METHODS: Adult male Sprague-Dawley rats were randomly treated with a single intracranial dose of lactacystin (2 or 5 µg) or saline (served as the sham control) into the left medial forebrain bundle. A 30-min static [18F]FP-(+)-DTBZ brain PET scan was performed following an intravenous [18F]FP-(+)-DTBZ dose (approximately 22 MBq) in each animal at 2 and 3 weeks after lactacystin treatment. Upon completing the last PET scans, the animals were killed, and their brains were dissected for ex vivo autoradiography (ARG) and immunohistochemical (IHC) staining of tyrosine hydroxylase (TH) as well as VMAT2. RESULTS: Both the 2- and 5-µg lactacystin-treated groups exhibited significantly decreased specific [18F]FP-(+)-DTBZ uptake in the ipsilateral striata (I-ST) at 2 weeks (1.51 and 1.16, respectively) and 3 weeks (1.36 and 1.00, respectively) after lactacystin treatment, compared with the uptake in the corresponding contralateral striata (C-ST) (3.48 and 3.08 for the 2- and 5-µg lactacystin-treated groups, respectively, at 2 weeks; 3.36 and 3.11 for the 2- and 5-µg lactacystin-treated groups, respectively, at 3 weeks) and the sham controls (3.34-3.53). Lactacystin-induced decline in I-ST [18F]FP-(+)-DTBZ uptake was also demonstrated through ex vivo ARG, and the corresponding dopaminergic neuron damage was confirmed by the results of TH- and VMAT2-IHC studies. CONCLUSIONS: In this PD model, lactacystin-induced dopaminergic terminal damage in the ipsilateral striatum could be clearly visualized through in vivo [18F]FP-(+)-DTBZ PET imaging. This may serve as a useful approach for evaluating the effectiveness of new treatments for PD.
Assuntos
Acetilcisteína/análogos & derivados , Doença de Parkinson/diagnóstico por imagem , Tetrabenazina/análogos & derivados , Acetilcisteína/farmacologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Radioisótopos de Flúor/metabolismo , Processamento de Imagem Assistida por Computador , Masculino , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Ratos , Ratos Sprague-Dawley , Tetrabenazina/metabolismoRESUMO
Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H+ per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen- to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H+-coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation.
Assuntos
Mutação , Reserpina/metabolismo , Tetrabenazina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/genética , Animais , Sítios de Ligação , Citoplasma/genética , Citoplasma/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Prótons , Ratos , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
OBJECTIVE: Cerebrospinal fluid (CSF) biomarkers, such as α-synuclein (α-syn), amyloid beta peptide 1-42 (Aß1-42), phosphorylated tau (181P) (p-tau), and total tau (t-tau), have long been associated with the development of Parkinson disease (PD) and other neurodegenerative diseases. In this investigation, we reported the assessment of CSF biomarkers and their correlations with vesicular monoamine transporter 2 (VMAT2) bindings measured with 18F-9-fluoropropyl-(+)-dihydrotetrabenazine (18F-AV133) that is being developed as a biomarker for PD. We test the hypothesis that monoaminergic degeneration was correlated with CSF biomarker levels in untreated PD patients. METHODS: The available online data from the Parkinson's Progression Markers Initiative study (PPMI) project were collected and analyzed, which include demographic information, clinical evaluations, CSF biomarkers (α-syn, Aß1-42, p-tau, and t-tau), 18F-AV133 brain PET, and T1 weighted MRIs. Region of interest (ROI) and voxel-wise Pearson correlation between standardized uptake value ratio (SUVR) and CSF biomarkers were calculated. RESULTS: Our major findings are: 1) Compared with controls, CSF α-syn and tau levels decreased significantly in PD; 2) α-syn was closely correlated with Aß1-42 and tau in PD, especially in early-onset patients; and 3) hypothesis-driven ROI analysis found a significant negative correlation between CSF Aß1-42 levels and VMAT2 densities in post cingulate, left caudate, left anterior putamen, and left ventral striatum in PDs. CSF t-tau and p-tau levels were significantly negatively related to VMAT2 SUVRs in substantia nigra and left ventral striatum, respectively. Voxel-wise analysis showed that left caudate, parahippocampal gyrus, insula and temporal lobe were negatively correlated with Aß1-42. In addition, superior frontal gyrus and transverse temporal gyrus were negatively correlated with CSF p-tau levels. CONCLUSION: These results suggest that monoaminergic degeneration in PD is correlated with CSF biomarkers associated with cognitive impairment in neurodegenerative diseases including Alzheimer's disease. The association between loss of dopamine synaptic function and pathologic protein accumulations in PD indicates an important role of CSF biomarkers in PD development.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Encéfalo/diagnóstico por imagem , Doença de Parkinson/diagnóstico por imagem , Tetrabenazina/análogos & derivados , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Idoso , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Encéfalo/metabolismo , Feminino , Radioisótopos de Flúor/metabolismo , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/metabolismo , Projetos Piloto , Tomografia por Emissão de Pósitrons , Tetrabenazina/metabolismo , alfa-Sinucleína/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidianoRESUMO
INTRODUCTION: 18F-Fluoropropyl-(+)-dihydrotetrabenazine (18F-FP-(+)-DTBZ) is a vesicular monoamine transporter type 2 (VMAT2) radiotracer for positron emission tomography (PET) imaging to quantify human ß-cell mass. Renal cortex and spleen have been suggested as reference regions, however, little is known about 18F-FP-(+)-DTBZ binding in these regions including the fraction of radiometabolite. We compared the kinetics of 18F-FP-(+)-DTBZ and its inactive enantiomer 18F-FP-(-)-DTBZ in baboons, estimated the non-displaceable binding (VND) of the tracers, and used ex vivo studies to measure radiometabolite fractions. METHODS: PET scans were conducted for up to 4h with (+) and (-) enantiomers. Displacement experiments using unlabeled (+) and (-) enantiomers of FP-DTBZ and fluvoxamine (to evaluate sigma-1 receptor binding) were performed. SUV curves were used to calculate displacement values in the pancreas, renal cortex, and spleen. Distribution volumes (VT) were computed, and three approaches for calculation of VND were compared: (1) 18F-FP-(+)-DTBZ reference VT, (2) 18F-FP-(-)-DTBZ pancreatic VT, and (3) a scaled 18F-FP-(+)-DTBZ reference VT values. Ex vivo study was conducted to measure radiometabolite fraction in homogenized tissue samples from baboons at 90min post-injection. RESULTS: Spleen uptake was lowest for both tracers. Highest uptake was in the pancreas with 18F-FP-(+)-DTBZ and renal cortex with 18F-FP-(-)-DTBZ. Substantial displacement effect was observed only with unlabeled FP-(+)-DTBZ in the 18F-FP-(+)-DTBZ studies. Radiometabolite fraction was higher in the renal cortex than the spleen. Approaches (1) and (3) with spleen to estimate VND provided lowest inter-subject variability of BPND. CONCLUSIONS: VT differences among organs and between enantiomers indicated that scaling of reference region values is needed for quantification of VMAT2 binding in the pancreas with 18F-FP-(+)-DTBZ. Since the kidney PET signal has greater partial volume averaging and more radiometabolites, the spleen was considered a more practical candidate for use as a scaled-reference region in the quantification of 18F-FP-(+)-DTBZ in the pancreas.
Assuntos
Pâncreas/metabolismo , Tetrabenazina/análogos & derivados , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Feminino , Radioisótopos de Flúor/química , Radioisótopos de Flúor/metabolismo , Cinética , Papio , Ligação Proteica , Estereoisomerismo , Tetrabenazina/química , Tetrabenazina/metabolismoRESUMO
A new capillary electrophoresis method for the enantioselective analysis of cis- and trans- dihydrotetrabenazine (diHTBZ) after in vitro metabolism by human liver microsomes (HLMs) was developed. The chiral electrophoretic separations were performed by using tris-phosphate buffer (pH 2.5) containing 1% (w/v) carboxymethyl-ß-CD as background electrolyte with an applied voltage of +15kV and capillary temperature kept at 15°C. Dispersive liquid-liquid microextraction was employed to extract the analytes from HLMs. Dichloromethane was used as extraction solvent (75µL) and acetone as disperser solvent (150µL). The method was validated according to official guidelines and showed to be linear over the concentration range of 0.29-19.57µmolL(-1) (r=0.9955) for each metabolite enantiomer. Within- and between-day precision and accuracy evaluated by relative standard deviation and relative error were lower than 15% for all enantiomers. The stability assay showed that the analytes kept stable under handling, storage and in metabolism conditions. After method validation, an enantioselective in vitro metabolism and in vivo pharmacokinetic prediction was carried out. This study showed a stereoselective metabolism and the observed kinetic profile indicated a substrate inhibition behavior. DiHTBZ enantiomers were catalyzed mainly by CYP2C19 and the predicted clearance suggests that liver metabolism is the main route for TBZ elimination which supports the literature data.
Assuntos
Eletroforese Capilar/métodos , Microextração em Fase Líquida/métodos , Microssomos Hepáticos/metabolismo , Tetrabenazina/farmacocinética , Citocromo P-450 CYP2C19/metabolismo , Estabilidade de Medicamentos , Humanos , Técnicas In Vitro , Reprodutibilidade dos Testes , Estereoisomerismo , Tetrabenazina/análogos & derivados , Tetrabenazina/metabolismoRESUMO
Our research groups recently described a series of small-molecule inducers of ß-cell proliferation that could be used to increase ß-cell mass. To mitigate the risk of nonspecific proliferation of other cell types, we devised a delivery strategy built on the tissue specificity observed in the experimental ß-cell imaging agent (+)-dihydrotetrabenazine (DTBZ). The ß-cell proliferator agent aminopyrazine (AP) was covalently linked with (+)-DTBZ to afford conjugates that retain both the proliferation activity and binding affinity for vesicular monoamine transporter-2 (VMAT2). Inâ vivo mouse tissue distribution studies of a prototypical AP-DTBZ conjugate showed 15-fold pancreas exposure over plasma. Tissue-to-plasma ratios in liver and kidneys were two- and five-fold, respectively. This work is the first demonstration of enhanced delivery of ß-cell-proliferating molecules to the pancreas by leveraging the intrinsic tissue specificity of a ß-cell imaging agent.
Assuntos
Pâncreas/metabolismo , Tetrabenazina/análogos & derivados , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Portadores de Fármacos/química , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Piridazinas/química , Coelhos , Espectrometria de Massas em Tandem , Tetrabenazina/sangue , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Distribuição Tecidual , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Pluripotent stem cells are considered as a cell source for replacement therapies for pancreatic beta cells and other organs.We identified tetrabenazine (TBZ), vesicular monoamine transporter 2 (VMAT2) inhibitor as a promoter of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Ngn3-positive endocrine progenitor cells. A cell-permeable cAMP analog, dBu-cAMP promotes beta cell maturation in late stage of differentiation. The induced beta cells can secrete insulin in a glucose-dependent manner.Our protocol consists of a three -step differentiation process. ES cell recapitulate embryonic developmental processes in vitro. Therefore, the ES cell differentiation system is a useful model for the understanding of molecular mechanism of beta-cell differentiation and are useful for application for future regenerative medicine.