Repositioning Antibiotics Against Plasmodium falciparum RAD5 and WD11 as Novel Antimalarial Targets

BACKGROUND: The emergence of artemisinin resistance in Plasmodium falciparum threatens the sustainability of malaria control programs and underscores the need for new therapeutic strategies. Recently, two proteins, RAD5 (PfRAD5) and WD40-repeat protein 11 (PfWD11), have been implicated in parasite survival and drug resistance. Their druggability, however, remains underexplored. METHODS: We integrated computational and experimental approaches to assess PfRAD5 and PfWD11 as antimalarial targets. Structural models were retrieved from AlphaFold and aligned with orthologues across Plasmodium species. A library of 216 antibiotics was screened by molecular docking, and molecular dynamics simulations. Top-ranked compounds were tested against P. falciparum 3D7 cultures in vitro, alone and in combination, and gene expression changes in PfRAD5 and PfWD11 were quantified by qPCR. Docking identified strong binders to PfRAD5 (talampicillin, dicloxacillin, raltegravir) and PfWD11 (cervinomycin A2 monoacetate, eAmSPC 2593, puromycin). RESULTS: Molecular dynamics confirmed the stability of protein-ligand complexes. In vitro, puromycin exhibited the highest inhibition (85% at 100 μg/mL), while combinations enhanced activity. The triple combination (puromycin + raltegravir + dicloxacillin) achieved complete inhibition, and puromycin + chloroquine exhibited synergistic effects at in a concentration-dependent manner. qPCR showed consistent downregulation of PfRAD5 and PfWD11 following puromycin-based treatments, whereas chloroquine alone upregulated PfRAD5 expression. PfRAD5 and PfWD11 represent novel antimalarial targets. CONCLUSION: Repurposed antibiotics, particularly puromycin in synergistic regimens, offer a cost-effective strategy to counteract emerging resistance and accelerate therapeutic development.