Abstract
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.
Highlights
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas’ disease, which is endemic in Latin American countries
The first gene copy of NMT could be successfully replaced with either hygromycin (HYG) or puromycin (PAC) resistance genes resulting in two independent single knockout (SKO) cell lines (Figures 1B, lane 3, and 1C, lane 2)
Previous studies have shown that the enzyme encoded by the NMT gene is essential for the survival of many eukaryotic organisms [10,12,35,36], including the related trypanosomatids L. major and T. brucei [12]
Summary
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas’ disease, which is endemic in Latin American countries. Benznidazole and nifurtimox are the only approved drugs available for the treatment of Chagas’ disease. Prolonged treatment with these nitroimidazoles during the acute stage cures up to 70 % of individuals; the efficacy of these drugs significantly decreases in the chronic stage [5]. Both therapies have been associated with severe toxic side effects that can lead to the interruption or discontinuation of treatment in as many as 30 % of cases [6,7]. Bearing in mind the high levels of drug candidate attrition in the clinical trials process, there remains an urgent need to identify new drug targets and better drugs to treat this disease
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