Mwitari, P. | Search bioRxiv

bioRxiv2019

Efficacy of Lumefantrine against piperaquine resistant Plasmodium berghei parasites is selectively restored by probenecid, verapamil, and cyproheptadine through ferredoxin NADP+-reductase and cysteine desulfurase

Chemotherapy remains central in the control of malaria; however, resistance has consistently thwarted these efforts. Currently, lumefantrine (LM), and piperaquine (PQ) drugs, are essential components in the mainstay artemisinin-based therapies used for the treatment of malaria globally. Using LM and PQ-resistant Plasmodium berghei, we measured the effect of known chemosensitizers: probenecid, verapamil, or cyproheptadine on the activity of LM or PQ. Using PlasmoGEM vectors, we then evaluated the impact of deleting cysteine desulfurase (SufS) or over-expressing Ferredoxin NADP+ reductase (FNR), genes that mediate drug action. Our data showed that, only cyproheptadine at 5mgkg-1 restored LM activity by above 65% against the LM-resistant parasites (LMR) but failed to reinstate PQ activity against the PQ-resistant parasites (PQR). Whereas the PQR had lost significant susceptibility to LM, the three chemosensitizers; cyproheptadine, probenecid, and verapamil, restored LM potency against the PQR by above 70%, 60%, and 55% respectively. We thus focused on LM resistance in PQR. Deletion of the SufS or overexpression of the FNR genes in the PQR abolished the impact of the chemosensitizers on the LM activity, and restored the susceptibility of the PQR parasites to LM. Taken together, we demonstrate the association between SufS or FNR genes with the action of LM and chemosensitizers in PQR parasites. There is, however, need to interrogate the impact of the chemosensitizers and the role of SufS or FNR genes on LM action in the human malaria parasite, Plasmodium falciparum.

genomics↗

bioRxiv2018

Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei

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.

molecular biology↗