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1.
J Biomol Screen ; 19(4): 595-605, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24241710

RESUMO

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.


Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Lipase Lipoproteica/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Lipase Lipoproteica/metabolismo , Reprodutibilidade dos Testes , Especificidade por Substrato
2.
Bioorg Med Chem Lett ; 23(6): 1684-8, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23414838

RESUMO

High throughput screening led to the identification of a novel series of quinolone α7 nicotinic acetylcholine receptor (nAChR) agonists. Optimization of an HTS hit (1) led to 4-phenyl-1-(quinuclidin-3-ylmethyl)quinolin-2(1H)-one, which was found to be potent and selective. Poor brain penetrance in this series was attributed to transporter-mediated efflux, which was in turn due to high pKa. A novel 4-fluoroquinuclidine significantly lowered the pKa of the quinuclidine moiety, reducing efflux as measured by a Caco-2 assay.


Assuntos
Agonistas Nicotínicos/química , Quinolonas/química , Receptores Nicotínicos/química , Animais , Células CACO-2 , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Agonistas Nicotínicos/síntese química , Agonistas Nicotínicos/metabolismo , Quinolonas/síntese química , Quinolonas/metabolismo , Ratos , Receptores Nicotínicos/metabolismo , Relação Estrutura-Atividade , Receptor Nicotínico de Acetilcolina alfa7
3.
Biochem Biophys Res Commun ; 411(4): 809-14, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21787747

RESUMO

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.


Assuntos
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
4.
J Biol Chem ; 282(51): 36829-36, 2007 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-17932033

RESUMO

The enzyme gamma-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of gamma-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and gamma-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid beta-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, gamma-secretase inhibitor radioligands were used to evaluate SPP and gamma-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for gamma-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of gamma-secretase binding sites, making it essential to use selective radioligands for evaluation of gamma-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Presenilinas/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Sulindaco/análogos & derivados , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Humanos , Ligantes , Modelos Moleculares , Presenilinas/metabolismo , Sulindaco/farmacologia
5.
Bioorg Med Chem Lett ; 17(14): 4006-11, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17502137

RESUMO

We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.


Assuntos
Alanina/análogos & derivados , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Benzodiazepinonas/farmacologia , Inibidores Enzimáticos/farmacologia , Alanina/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides , Animais , Camundongos , Camundongos Transgênicos , Modelos Moleculares
6.
Anal Biochem ; 349(1): 112-7, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16325755

RESUMO

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.


Assuntos
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
7.
J Pharmacol Exp Ther ; 307(2): 682-91, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12975483

RESUMO

Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.


Assuntos
Aprendizagem/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Sítios de Ligação , Humanos , Mastigação/efeitos dos fármacos , Camundongos , Modelos Animais , Pirimidinas/farmacologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Serotonina/efeitos dos fármacos , Bocejo/efeitos dos fármacos
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