ResMAP – a saturation mutagenesis platform enabling parallel profiling of target-specific resistance conferring mutations in Plasmodium

New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance, thus new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a resistance mapping and profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, P. falciparum lysyl tRNA synthetase (PfKRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20 residue span at the base of the PfKRS ATP binding pocket. The superior transfection efficiency of P. knowlesi was exploited to generate a high coverage parasite library expressing PfKRS bearing all possible amino acid changes within this region of the enzyme. Selection of the library with PfKRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modelling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed.