Authors

Discussion

The complex life cycle and extensive genetic diversity of P. falciparum pose significant challenges for antimalarial drug development, particularly in the context of emerging partial resistance to artemisinin-based therapies. Although the scale of antimalarial screening and discovery has increased over the past two decades, identifying the molecular targets of novel compounds remains time-consuming and resource-intensive. Conventional approaches for target identification typically involve cross-resistance testing against parasite lines carrying known resistance mutations, proteomics-based techniques, or prolonged in vitro resistance selection followed by whole-genome sequencing and genetic validation. To accelerate this process, we developed the Antimalarial Resistome Barcode assay (AReBar), a genome-editing–enabled cross-resistance screening platform that allows rapid and comprehensive compound profiling. The current AReBar pool consists of 52 uniquely barcoded parasite lines representing a broad spectrum of validated drug targets and resistance mechanisms. Here, we report the screening results of over 200 compounds with unknown modes of action. Among these, 31 compounds exhibited cross-resistance in parasite lines carrying mutations in established targets, including PfPI4K, PfATP4, PfDHODH, PfMCP, PfCytBC1, PfProteasome β5, and PfCRT. In parallel, methodological refinements were explored; a ramping drug-selection protocol over nine days yielded promising results, although further optimization is warranted. Overall, AReBar provides a rapid and reliable platform for identifying potential cross-resistance patterns, thereby facilitating mechanism-of-action studies and accelerating antimalarial drug discovery.