Deciphering the targets of 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 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.\n\nAuthor summaryThe antiretroviral drugs (ARVs) such as LP or SQ that inhibit viral proteases reduce the rate of multiplication of the malaria parasites. The mode of action of these drugs against the parasites is however poorly understood. The proteases are among the enzymes that play essential roles in Plasmodium parasites. We sought to investigate the possible mode of action of these drugs by generating mutant parasites lacking specific aspartyl proteases namely PM4, PM7, PM8 or Ddi1 and then evaluate the susceptibility of the mutants to LP and SQ. We successfully generated parasites lacking either PM4, PM7 or PM8 but Ddi1 gene was refractory to deletion. From our data, we demonstrate that, unlike PM7 and PM8, the PM4 and Ddi1 are essential enzymes for asexual blood stage parasite fitness and survival and that the PM4 might be a target for the viral protease inhibitors in reducing parasite growth and multiplication. Further experiments using molecular docking tools show that LP or SQ have a high binding affinity for the Plasmodium aspartyl proteases.