RESUMO
N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex.
Assuntos
Aciltransferases/antagonistas & inibidores , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , Peptidomiméticos/farmacologia , Plasmodium/enzimologia , Aciltransferases/metabolismo , Antifúngicos/síntese química , Antifúngicos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Leishmania/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Peptidomiméticos/síntese química , Peptidomiméticos/química , Plasmodium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.