Abstract
Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.
Highlights
Notwithstanding the immense investments in malaria control programs to date, it remains to be a significant global health problem in most regions of the world including Africa, Asia and parts of the Eastern Mediterranean Region [1,2]
Generation of the Plasmepsin 4 (PM4), PM7, PM8 or damage-inducible protein 1 (Ddi1) knockout parasites: The Ddi1 gene was refractory to deletion essential for the growth of the asexual blood stage parasites
Three successive attempts to delete the Ddi1 failed to recover parasite twenty days post infection suggesting that the Ddi1 gene was refractory to deletion and essential for the asexual blood stage parasite growth
Summary
Notwithstanding the immense investments in malaria control programs to date, it remains to be a significant global health problem in most regions of the world including Africa, Asia and parts of the Eastern Mediterranean Region [1,2]. The burden of malaria is compounded with HIV/AIDS infections which are concentrated in the malaria-endemic regions, primarily sub-Saharan Africa. This geographical overlap has raised opportunities and concerns for potential immunological, social, therapeutic and clinical interactions [7]. The PM5, PM9 and PM10 are the other aspartyl proteases expressed within asexual blood stage of the malaria parasite but not localised within the digestive vacuole [24]. We show the in silico binding profiles of LP and SQ on the Ddi protein and provide evidence that the retropepsin-like protease is essential for the asexual stage of the malaria parasite
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