RESUMEN
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.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antifúngicos/farmacología , Inhibidores Enzimáticos/farmacología , Leishmania/enzimología , Peptidomiméticos/farmacología , Plasmodium/enzimología , Aciltransferasas/metabolismo , Antifúngicos/síntesis química , Antifúngicos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Leishmania/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Plasmodium/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
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.