Biochemical and genetic characterization of Trypanosoma cruzi N-myristoyltransferase.
Co- and post-translational N-myristoylation is known to play a role in the correct subcellular localization of specific proteins in eukaryotes. The enzyme that catalyses this reaction, NMT (N-myristoyltransferase), has been pharmacologically validated as a drug target in the African trypanosome, Trypanosoma brucei. In the present study, we evaluate NMT as a potential drug target in Trypanosoma cruzi, the causative agent of Chagas' disease, using chemical and genetic approaches. Replacement of both allelic copies of TcNMT (T. cruzi NMT) was only possible in the presence of a constitutively expressed ectopic copy of the gene, indicating that this gene is essential for survival of T. cruzi epimastigotes. The pyrazole sulphonamide NMT inhibitor DDD85646 is 13-23-fold less potent against recombinant TcNMT than TbNMT (T. brucei NMT), with Ki values of 12.7 and 22.8 nM respectively, by scintillation proximity or coupled assay methods. DDD85646 also inhibits growth of T. cruzi epimastigotes (EC50=6.9 μM), but is ~1000-fold less potent than that reported for T. brucei. On-target activity is demonstrated by shifts in cell potency in lines that over- and under-express NMT and by inhibition of intracellular N-myristoylation of several proteins in a dose-dependent manner. Collectively, our findings suggest that N-myristoylation is an essential and druggable target in T. cruzi.
*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Adam J
AJ
Roberts
*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Leah S
LS
Torrie
*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Susan
S
Wyllie
*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Alan H
AH
Fairlamb
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000631:Aminopyridines; D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007700:Kinetics; D030781:Organisms, Genetically Modified; D011994:Recombinant Proteins; D013449:Sulfonamides; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014709:Vero Cells; D000096926:Benzenesulfonamides
D000217:Acyltransferases; D000631:Aminopyridines; D011994:Recombinant Proteins; D013449:Sulfonamides; C549249:DDD 85646; D000096926:Benzenesulfonamides; C050480:glycylpeptide N-tetradecanoyltransferase