G-quadruplex-binding ligand RHPS4 reduces Plasmodium falciparum mitochondrial DNA level providing a novel antimalarial strategy.

Malaria caused by Plasmodium falciparum remains a major health threat, killing over 600,000 people annually. The spread of resistance to all major antimalarials, including artemisinins, highlights the urgent need for new drugs with distinct mechanisms of action. Here, we show that the G-quadruplex ligand RHPS4, an acridine derivative, displays strong antiplasmodial activity against both drug-sensitive and drug-resistant P. falciparum strains and clinical isolates. RHPS4 primarily targets the trophozoite stage and induces major mitochondrial alterations, including reduction of mitochondrial DNA (mtDNA) and transcriptional dysfunctions. Bioinformatic analyses identified at least eight putative G4-forming sequences within the parasite's mtDNA. Biophysical studies confirmed G4 folding of at least one sequence and its interaction with RHPS4. These findings indicate that RHPS4 impacts P. falciparum mitochondrial DNA replication and transcription, leading to parasite death, and establish G4 ligands as novel antimalarial agents.