Sequencing MMV020291-resistant P. falciparum genomes to uncover the molecular targets of the compound

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell invasion, and have generated analogues with improved potency. Here, we identify actin and profilin as putative targets of the MMV020291 series through resistance selection and whole genome sequencing of three MMV020291 resistant populations. This revealed three non-synonymous single nucleotide polymorphisms in two genes; two the profilin gene (N154Y, K124N) and the third one in the actin-1 gene (M356L). Using CRISPR-Cas9, we engineered these mutations into wildtype parasites, reproducing parasite resistance to MMV020291. We demonstrate that MMV020291 blocks actin polymerisation that is required by the merozoite stage parasites to invade red blood cells. Additionally, the series inhibits the actin-1 dependent process of apicoplast segregation, leading to a delayed death phenotype. In in vitro co-sedimentation experiments, using P. falciparum actin-1 and profilin, potent MMV020291 analogues amplify the actin-monomer sequestering effect of profilin, indicating stabilization of the protein complex. Altogether, this study identifies the first compound series targeting the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.