Objective

Protein N-myristoylation is the attachment of the 14-carbon fatty acid myristate onto the N-terminal glycine of certain proteins. This largely co-translational modification is catalysed by N-myristoyltransferase (NMT) and is involved in localizing the substrate protein to specific membranes. In Plasmodium falciparum, NMT substrates are involved in a variety of essential processes including host cell invasion, parasite gliding motility, protein trafficking and protein degradation. NMT has been validated as a therapeutic target in numerous parasitic diseases including malaria. Treatment with an NMT inhibitor results in rapid reduction of P. falciparum growth in vitro and in vivo using a SCID mouse model. We have used crystallography to study the structural basis of potent NMT inhibitors selective for Plasmodium spp. with the enzyme. Parasites resistant to an entire inhibitor series were generated and we identified that resistance is mediated by a single amino acid substitution in the substrate binding pocket of NMT. The binding of another drug series remains unaffected by this mutation. We successfully manipulated P. falciparum using CRISPR-Cas9 to confirm that the G386E mutation selected under drug pressure was causal for in vitro resistance. We tagged myristoylated proteins expressed in the mutant parasite via a copper catalysed cycloaddition reaction (“click” chemistry) and multifunctional probes. We then introduced the same amino acid into recombinant Plasmodium vivax NMT, which can be easily expressed and purified. In vitro parasite growth-, enzyme activity and SPR assays with structurally different NMT inhibitors provided insight into the different drug binding modes. This approach – to incorporate the study of resistance early-on in the drug development process – will help formulation of solutions to bypass evolution of parasite drug resistance.