Repurposed small-molecule inhibitors of Plasmodium falciparum calmodulins disrupt calcium homeostasis.

Calcium signaling is essential for the survival of Plasmodium falciparum, yet few components of its Ca2+ regulatory machinery have been chemically validated as antimalarial drug targets. Here, we report the structure-guided identification of small molecule inhibitors targeting a parasite specific subset of calmodulin and calmodulin-like proteins that lack close human orthologs. Virtual screening followed by biophysical validation revealed that the drugs Econazole, Carvedilol, and Deschloro-Lapatinib selectively engage three parasite calmodulin paralogs (PfCam200, PfCam300, and PfCam400) and inhibit their Ca2+-binding activity. Direct binding was confirmed by bio-layer interferometry and calcium-binding assays, demonstrating micromolar affinities and functional inhibition. Chemical perturbation of parasite calmodulins disrupts food vacuole directed Ca2+ trafficking, induces redistribution of intracellular Ca2+, and results in stage specific arrest of parasite development. Confocal imaging localizes PfCam200, PfCam300, and PfCam400 in proximity to the digestive vacuole transporters PfCRT and PfMDR1, supporting a mechanistic link between calmodulin function and vacuolar Ca2+ homeostasis. Collectively, these findings establish parasite calmodulins as chemically tractable targets and demonstrate the utility of repurposed small molecules as probes to interrogate essential Ca2+-dependent pathways in malaria parasites.