Rhomboid Proteases in Plasmodium spp. : Biology, Functional Roles, and Therapeutic Potential

Malaria continues to rank among the most significant and deadly infectious diseases, with over 282 million estimated cases and more than 610,000 deaths reported by the WHO in 2025. With the continuous rise of resistance to currently existing antimalarial drugs, there is a dire need to validate new drug targets. Evolutionarily conserved across eukaryotes, rhomboid proteases constitute a family of intramembrane serine proteases that play essential roles in parasite development, motility, and host cell invasion. This review summarizes the biology, structure, catalytic mechanism, and substrate specificity of Plasmodium rhomboid proteases, and critically evaluates their functional roles in parasite development, host-cell invasion, and potential as antimalarial drug targets.

The available literature on Plasmodium rhomboid protease biology, structural architecture, catalytic mechanism, substrate specificity, genetic essentiality, and emerging rhomboid-directed antimalarial therapeutic strategies was reviewed in a narrative format.

Among the eight Plasmodium rhomboid proteases, PfROM1 and PfROM4 are the best characterized, with PfROM4 serving as the main protease responsible for shedding key invasion-related adhesins (EBL, RBL, and MTRAP families) during erythrocyte invasion. Systematic knockout experiments demonstrated that PbROM4, PbROM6, and PbROM7 could not be deleted, establishing their essentiality in that species; however, recent inducible knockout studies have independently confirmed essentiality for PfROM4, PfROM6, and PfROM8 directly in P. falciparum. Parasite-selective inhibitors of PfROM4, including a peptide boronate and an α-ketoamide, have demonstrated the ability to block erythrocyte invasion and clear blood-stage parasitemia, providing the first proof of concept for rhomboid-targeted antimalarial therapy.

Most Plasmodium rhomboid proteases remain partially characterized or uncharacterized, and no experimental structure of a Plasmodium rhomboid has yet been solved. Recent development of parasite-selective inhibitors for PfROM4 offers an encouraging early proof of concept that this protease family can be pharmacologically targeted; however, this research was performed in vitro, and further investigation in animal models and against human rhomboids is required.