ABSTRACT
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.
Subject(s)
Acyltransferases/antagonists & inhibitors , Antifungal Agents/pharmacology , Enzyme Inhibitors/pharmacology , Leishmania/enzymology , Peptidomimetics/pharmacology , Plasmodium/enzymology , Acyltransferases/metabolism , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Leishmania/drug effects , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Peptidomimetics/chemical synthesis , Peptidomimetics/chemistry , Plasmodium/drug effects , Structure-Activity RelationshipABSTRACT
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.
