Chemoproteomics-based profiling elucidates the antimalarial effects of amodiaquine through disruption of glycolysis process in Plasmodium falciparum

BACKGROUND: Malaria poses a significant global health threat, and 4-aminoquinolines have played a pivotal role in the decades-long fight against malaria. Amodiaquine (AQ), a prominent member of the class, has been employed clinically for decades and, in combination with artesunate (AS), constitutes one of the most widely applied artemisinin-based combination therapies (ACTs). However, the precise molecular targets and antimalarial mechanisms of AQ remain incompletely understood. METHODS: We synthesized an AQ-derived activity probe (AQP) and systematically identified AQ-binding proteins using an activity-based protein profiling (ABPP) strategy. Integrative proteomic and transcriptomic analyses were then performed to characterize the pathways and potential targets associated with AQ action. RESULTS: We identified three glycolysis-associated enzymes as potential antimalarial targets of AQ. Subsequent validation experiments confirmed that AQ binds to these proteins and disrupts glycolytic processes in Plasmodium falciparum (P. falciparum). Moreover, we investigated the interactions between AS and AQ and demonstrated their complementary effects on shared molecular targets, suggesting a potential mechanism underlying the enhanced efficacy of AS-AQ combination therapy. CONLUSIONS: Our findings reveal that AQ exerts its antimalarial effects by binding to key glycolytic enzymes in P. falciparum and highlight the interplay between AQ and AS in targeting parasite metabolism. This work deepens the mechanistic understanding of AQ and AS-AQ, providing new insights into the mode of action of ACTs and offering potential strategies for future antimalarial drug development.