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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
J Biomol Screen ; 15(1): 72-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19959816


Deoxycytidine kinase (dCK) phosphorylates deoxycytidine, deoxyguanosine, and deoxyadenosine and plays an important role in the salvage pathway of nucleoside metabolism. dCK is also required for the phosphorylation of several antiviral and anticancer nucleoside drugs, with resistance to these agents often being associated with a loss or decrease in dCK activity. Data also indicate a role for dCK in immune function, and dCK inhibitors may provide treatment for immune disorders. To identify novel dCK inhibitors, the authors evaluated 2 existing biochemical assays, adapted both to high-throughput screening, and identified several series of hits. They also compared the potency of the hits between purified recombinant and endogenous enzyme. Meanwhile, they also developed a novel cell-based assay that rests on the rescue of cells from dCK-dependent cytotoxic agents such as AraC. A large number of compounds were tested using the 3 assays, and a strong correlation in potency was observed between the biochemical assay using endogenous enzyme and the cell-based assay. The hits identified in these screens have proved to be good starting points for the synthesis of much more potent tool compounds to further investigate the physiological functions of dCK and potentially lead to the development of therapeutic agents.

Desoxicitidina Quinase/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Morte Celular/efeitos dos fármacos , Humanos , Medições Luminescentes , Proteínas Recombinantes/antagonistas & inibidores , Fatores de Tempo
Bioorg Med Chem Lett ; 19(23): 6780-3, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19836229


A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.

Desoxicitidina Quinase/antagonistas & inibidores , Flucitosina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Desenho de Fármacos , Flucitosina/síntese química , Flucitosina/química , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estereoisomerismo , Relação Estrutura-Atividade
Bioorg Med Chem Lett ; 19(23): 6784-7, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19836232


A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.

Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Desoxicitidina/síntese química , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estereoisomerismo , Relação Estrutura-Atividade
J Biol Chem ; 284(3): 1419-24, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19028676


Two members of the angiopoietin-like family of proteins, ANGPTL3 and ANGPTL4, have been shown to play important roles in modulating lipoprotein metabolism in the body. Both proteins were found to suppress lipoprotein lipase (LPL) activity in vitro as well as in vivo. However, their mechanisms of inhibition remained poorly understood. Using enzyme kinetic analysis with purified recombinant proteins, we have found key mechanistic differences between ANGPTL3 and ANGPTL4. ANGPTL3 reduced LPL catalytic activity but did not significantly alter its self-inactivation rate. In contrast, ANGPTL4 suppressed LPL by accelerating the irreversible inactivation of LPL. Furthermore, heparin was able to overcome the inhibitory effect of ANGPTL3 on LPL but not that of ANGPTL4. Site-directed mutagenesis demonstrated the critical function of Glu(40) in ANGPTL4. In contrast, when cysteine residues involved in disulfide bond formation were mutated to serines, ANGPTL4 retained its activity. Taken together, our data provide a more detailed view of the structure and mechanisms of these proteins. The finding that ANGPTL3 and ANGPTL4 inhibit LPL activity through distinct mechanisms indicates that the two proteins play unique roles in modulation of lipid metabolism in vivo.

Angiopoietinas/química , Metabolismo dos Lipídeos/fisiologia , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/química , Proteína 4 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Angiopoietinas/metabolismo , Catálise , Linhagem Celular , Ativação Enzimática/fisiologia , Heparina/química , Humanos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade