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.
