Identification of an inhibitory pocket in falcilysin bound by chloroquine provides a new avenue for malaria drug development

Despite their widespread use, our understanding of how malaria drugs work remains limited. This includes chloroquine (CQ), the most successful antimalarial ever deployed. Here, we used MS-CETSA and dose-response transcriptional profiling to identify possible protein targets and mechanism of action (MOA) of CQ, as well as MK-4815, a malaria drug candidate with a proposed MOA similar to CQ. Both compounds bind falcilysin (FLN) and hemoglobin digestion was the key biological pathway affected, with distinct MOA profiles between CQ-sensitive and CQ-resistant parasites. We showed that CQ and MK-4815 inhibit FLN proteolytic activity, and using X-ray crystallography, that they occupy a hydrophobic pocket situated within the large peptide substrate binding cavity of FLN. Studies using transgenic parasite line suggest the potential role of FLN in the CQ and MK-4815 MOA. Altogether, our data reveal a druggable pocket in the FLN substrate binding cavity that can be explored in future antimalarial development efforts.