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1.
Nat Med ; 29(6): 1487-1499, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37291212

RESUMO

Cannabis use disorder (CUD) is widespread, and there is no pharmacotherapy to facilitate its treatment. AEF0117, the first of a new pharmacological class, is a signaling-specific inhibitor of the cannabinoid receptor 1 (CB1-SSi). AEF0117 selectively inhibits a subset of intracellular effects resulting from Δ9-tetrahydrocannabinol (THC) binding without modifying behavior per se. In mice and non-human primates, AEF0117 decreased cannabinoid self-administration and THC-related behavioral impairment without producing significant adverse effects. In single-ascending-dose (0.2 mg, 0.6 mg, 2 mg and 6 mg; n = 40) and multiple-ascending-dose (0.6 mg, 2 mg and 6 mg; n = 24) phase 1 trials, healthy volunteers were randomized to ascending-dose cohorts (n = 8 per cohort; 6:2 AEF0117 to placebo randomization). In both studies, AEF0117 was safe and well tolerated (primary outcome measurements). In a double-blind, placebo-controlled, crossover phase 2a trial, volunteers with CUD were randomized to two ascending-dose cohorts (0.06 mg, n = 14; 1 mg, n = 15). AEF0117 significantly reduced cannabis' positive subjective effects (primary outcome measurement, assessed by visual analog scales) by 19% (0.06 mg) and 38% (1 mg) compared to placebo (P < 0.04). AEF0117 (1 mg) also reduced cannabis self-administration (P < 0.05). In volunteers with CUD, AEF0117 was well tolerated and did not precipitate cannabis withdrawal. These data suggest that AEF0117 is a safe and potentially efficacious treatment for CUD.ClinicalTrials.gov identifiers: NCT03325595 , NCT03443895 and NCT03717272 .


Assuntos
Cannabis , Alucinógenos , Abuso de Maconha , Síndrome de Abstinência a Substâncias , Animais , Camundongos , Método Duplo-Cego , Dronabinol/efeitos adversos , Alucinógenos/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Síndrome de Abstinência a Substâncias/tratamento farmacológico
2.
J Neurosci Methods ; 332: 108543, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830543

RESUMO

BACKGROUND: Phosphorylation by protein kinases is a fundamental molecular process involved in the regulation of signaling activities in living organisms. Understanding this complex network of phosphorylation, especially phosphoproteins, is a necessary step for grasping the basis of cellular pathophysiology. Studying brain intracellular signaling is a particularly complex task due to the heterogeneous complex nature of the brain tissue, which consists of many embedded structures. NEW METHOD: Overcoming this degree of complexity requires a technology with a high throughput and economical in the amount of biological material used, so that a large number of signaling pathways may be analyzed in a large number of samples. We have turned to Alpha (Amplified Luminescent Proximity Homogeneous Assay) technology. COMPARISON WITH EXISTING METHOD: Western blot is certainly the most commonly used method to measure the phosphorylation state of proteins. Even though Western blot is an accurate and reliable method for analyzing modifications of proteins, it is a time-consuming and large amounts of samples are required. Those two parameters are critical when the goal of the research is to comprehend multi-signaling proteic events so as to analyze several targets from small brain areas. RESULT: Here we demonstrate that Alpha technology is particularly suitable for studying brain signaling pathways by allowing rapid, sensitive, reproducible and semi-quantitative detection of phosphoproteins from individual mouse brain tissue homogenates and from cell fractionation and synaptosomal preparations of mouse hippocampus. CONCLUSION: Alpha technology represents a major experimental step forward in unraveling the brain phosphoprotein-related molecular mechanisms involved in brain-related disorders.


Assuntos
Fosfoproteínas , Transdução de Sinais , Animais , Western Blotting , Encéfalo/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Fosforilação
3.
Cancers (Basel) ; 11(3)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832225

RESUMO

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101's action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

4.
Anal Chem ; 81(23): 9590-8, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19873978

RESUMO

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.


Assuntos
Corantes Fluorescentes/metabolismo , Espaço Intracelular/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proliferação de Células , Descoberta de Drogas , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Humanos , Imagem Molecular , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/química , Proteína X Associada a bcl-2/metabolismo
5.
J Cell Sci ; 121(Pt 21): 3515-23, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18840646

RESUMO

Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimers that are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homo-multimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca(2+) mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.


Assuntos
Apoptose , Citocromos c/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína X Associada a bcl-2/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Dimerização , Inativação Gênica , Células HeLa , Humanos , Mitocôndrias/metabolismo , Modelos Biológicos , Conformação Proteica
6.
Biochim Biophys Acta ; 1783(8): 1551-60, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18424271

RESUMO

gamma-Secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP), Notch and other cellular substrates and is considered a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). We describe here an efficient, new, simple, sensitive and rapid assay to quantify gamma-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for gamma-secretase. The principle of the assay is based on the fact that the soluble intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane into the cytosol following gamma-secretase cleavage. Using this feature, enzymatic activity of gamma-secretase could be deduced from the extent of the membrane retention of the probe observed after plasma membrane permeabilization and washout of the cleaved fraction. By applying two well-known gamma-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for gamma-secretase activity behave properly when expressed in different cell lines, providing the basis for the further development of a high-throughput and high content screening for AD targeted drug discovery. Moreover, by co-expression of different familial AD-linked mutated forms of presenilin--the key component of the gamma-secretase complex--in cells devoid of any endogenous gamma-secretase, our method allowed us to evaluate in situ the contribution of different presenilin variants to the modulation of the enzyme.


Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/análise , Citometria de Fluxo , Mutação , Presenilinas/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Proteínas Recombinantes/análise
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