A novel 2-piperazino-pyrimidine compound exhibits asexual antimalarial activity by targeting Plasmodium falciparum plasmepsin X.

Although the number of cases of malaria has declined since the introduction of artemisinin-based combination therapy in endemic countries, the emergence of resistant parasites in the field has led to an alarming increase in treatment failure, making the search for novel drug candidates with precise targets critical. Here, we report the identification, from Shionogi's chemical library, of a 2-piperazino-pyrimidine compound (FPSA hereafter), with antimalarial activity; the compound shows a Plasmodium falciparum-killing effect resulting from blockage of merozoite release from infected erythrocytes. Parasites treated with FPSA halted as schizonts and exhibited a defect in the egress process. After extended selection, we generated FPSA-resistant parasites and obtained five clones showing modest EC50 shifts. Whole-genome sequencing of these isolates identified 2 clones harboring distinct single-nucleotide variants in the plasmepsin X (PMX)-encoding gene (PfDd2_080013400), changes that were predicted to result in I252V or E55G substitutions in PMX; another 3 clones harbored duplications of the genomic region spanning PfDd2_080013400. Genome editing parasites harboring the corresponding amino acid substitutions exhibited EC50 fold changes similar to those observed in the resistant parasites. Furthermore, FPSA inhibited the proteolytic activity of recombinant PMX in vitro and also the cleavage of PMX substrate proteins in parasites. Transcriptomic analysis confirmed that parasites exposed to FPSA exhibit gene expression patterns similar to those observed in parasites exposed to WM382, a validated plasmepsin IX (PMIX)/PMX dual inhibitor. These results indicate that FPSA inhibits merozoite egress by targeting P. falciparum plasmepsin X. Therefore, this work identifies a novel class of antimalarial compounds that target PMX. Our results are expected to contribute to the development of new drugs for the treatment of malaria.