RESUMO
Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.
Assuntos
Fosfolipases A/antagonistas & inibidores , Pirimidinonas/química , Pirimidinonas/farmacologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Fosfolipases A2 , Pirimidinonas/síntese química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.