IDENTIFICATION OF POTENTIAL TARGETS FOR RETROVIRAL PROTEASE INHIBITORS IN Plasmodium berghei

Abstract

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact target(s) for these RPIs in the Plasmodium parasites is unknown. We hypothesized that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, the study embarked on separately generating transgenic parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. The suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) were then tested against the transgenic parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to the deletion, thus essential for the asexual blood stage parasites. Study results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.0032). Unlike PM4_KO parasites which were less sensitive to LP and SQ (P = 0.0364, P = 0.0303), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites (P = 0.938 - 0.559, P = 0.6634 - 0.2013). This finding suggests potential role of PM4 in the LP and SQ action. Further analysis using modelling and molecular docking studies revealed that both LP and SQ had high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. It was concluded that PM4 plays an important role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The indispensable nature of the newly identified Ddi1 gene warrants further studies to evaluate its role in parasite asexual stage survival as well as its candidature as a target for RPIs.