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Stem Cell Res ; 20: 67-69, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28395743


Human fibroblast cells collected from a 3-year old, female Rett Syndrome patient with a 32bp deletion in the X-linked MECP2 gene were obtained from the Coriell Institute. Fibroblasts were reprogrammed to iPSC cells using a Sendai-virus delivery system expressing human KOSM transcription factors. Cell-line pluripotency was demonstrated by gene expression, immunocytochemistry, in-vitro differentiation trilineage capacity and was of normal karyotype. Interestingly, subsequent clones retained the epigenetic memory of the parent fibroblasts allowing for the segregation of wild-type and mutant expressing clones. This MECP2 mutant expressing clone may serve as a model for investigating MECP2 reactivation in Rett's Syndrome.

Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/patologia , Alelos , Diferenciação Celular , Linhagem Celular , Pré-Escolar , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Vírus Sendai/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
J Pharmacol Exp Ther ; 354(3): 340-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26109678


The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

Regulação Alostérica/efeitos dos fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , Animais , Células CHO , Linhagem Celular , Células Cultivadas , Cricetulus , Células HEK293 , Humanos , Camundongos , Ratos , Esquizofrenia/tratamento farmacológico
J Neurochem ; 129(2): 275-83, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24266811


NMDA receptor hypofunction is hypothesized to contribute to cognitive deficits associated with schizophrenia. Since direct activation of NMDA receptors is associated with serious adverse effects, modulation of the NMDA co-agonists, glycine or D-serine, represents a viable alternative therapeutic approach. Indeed, clinical trials with glycine and D-serine have shown positive results, although concerns over toxicity related to the high-doses required for efficacy remain. Synaptic concentrations of D-serine and glycine are regulated by the amino acid transporter alanine serine cysteine transporter-1 (asc-1). Inhibition of asc-1 would increase synaptic D-serine and possibly glycine, eliminating the need for high-dose systemic D-serine or glycine treatment. In this manuscript, we characterize Compound 1 (BMS-466442), the first known small molecule inhibitor of asc-1. Compound 1 selectively inhibited asc-1 mediated D-serine uptake with nanomolar potency in multiple cellular systems. Moreover, Compound 1 inhibited asc-1 but was not a competitive substrate for this transporter. Compound 1 is the first reported selective inhibitor of the asc-1 transporter and may provide a new path for the development of asc-1 inhibitors for the treatment of schizophrenia.

Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Agonistas de Aminoácidos Excitatórios/farmacologia , Histidina/análogos & derivados , Indóis/síntese química , Indóis/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Aminoácidos/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Glicina/metabolismo , Histidina/síntese química , Histidina/farmacologia , Humanos , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Bibliotecas de Moléculas Pequenas , Sinaptossomos/metabolismo
Biochem Biophys Res Commun ; 411(4): 809-14, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21787747


Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.

Lipase Lipoproteica/química , Catálise , Membrana Celular/enzimologia , Compostos Cromogênicos/química , Cicloexanonas/química , Fluorescência , Células HEK293 , Humanos , Lactonas/química , Lipase Lipoproteica/genética , Mutação , Orlistate , Especificidade por Substrato
Mol Pharmacol ; 69(4): 1396-404, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16399850


Neurotrophins are a family of secreted proteins that play an important role in the development, differentiation, and survival of neurons. Studies also suggest that aberrant neurotrophin signaling may play a role in processes underlying disease states such as schizophrenia, Alzheimer's disease, and depression. Whereas the development of agents that selectively stimulate neurotrophin signaling has proven to be difficult, compounds have been identified that potentiate neurotrophin 3 (NT-3)-mediated activation of trk A. In the present studies, we extend those initial observations to identify compounds that also potentiate NT-3-mediated activation of trk B. Compound potentiation of NT-3 was observed using several readouts of transfected and endogenous trk receptor activity, including trk receptor phosphorylation, mitogen-activated protein kinase phosphorylation, reporter assay activity (beta-lactamase and luciferase), cell survival and neurite extension assays. Studies using chimeric trk receptors demonstrated that the extracellular domain is essential for compound potentiation and rule out interaction with intracellular signaling molecules as a mechanism of compound activity. Thus, the present studies demonstrate that trk B receptor activity can be potentiated by small-molecule compounds via the extracellular domain of the receptor and provide reagents for further evaluating the role of NT-3-mediated trk A and trk B activity in vivo.

Neurotrofina 3/farmacologia , Receptor trkB/agonistas , Animais , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , DNA Complementar , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Transdução de Sinais
Anal Biochem ; 349(1): 112-7, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16325755


Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.

Catecol O-Metiltransferase/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Catecol O-Metiltransferase/química , Catecol O-Metiltransferase/fisiologia , Ativação Enzimática , Humanos , Cinética , Dados de Sequência Molecular , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo