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.

Malaria parasite plasmepsins: More than just plain old degradative pepsins

“…It has only one catalytic aspartate and homodimerizes to form the active enzyme. In malaria parasites, all that is known is that its gene is refractory to knockout in P. berghei and P. falciparum (160,168,169).…”

“…The digestive vacuole plasmepsins, Ddi1, SPP, and hexose transporter PfHT1 have all been proposed (169,(184)(185)(186)(187)(188).…”

Toxoplasma gondii UBL‐UBA shuttle proteins contribute to the degradation of ubiquitinylated proteins and are important for synchronous cell division and virulence

“…47 Several studies have demonstrated that HIV proteinase inhibitors inhibit the growth of Plasmodium and Toxoplasma. [48][49][50] Hence, whether T gondii DDI1 was also the target of HIV proteinase inhibitors would be an interesting topic need to address.…”

Repurposing Drugs to Fight Hepatic Malaria Parasites

“…Lopinavir and ritonavir were further reported to reduce ookinete and oocyst formation by P. berghei , while ritonavir, saquinavir, and indinavir were shown to inhibit this parasite’s oocyst development [ 132 ]. The activity of PIs against Plasmodium parasites has been suggested to be mediated by the inhibition of their aspartyl proteases, some of which localize to the food vacuole and are involved in the degradation of haemoglobin [ 28 , 133 , 134 , 135 , 136 ]. The assessment of the activity of the NNRTIs efavirenz, etravirine, and nevirapine against the blood stage of P. falciparum revealed that the latter is not effective [ 31 , 34 ].…”