System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion

Abstract

Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a "multi-omics" workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonideinduced depletion of short Hb-derived peptides was less extensive in a drug-treated K13mutant artemisinin resistant parasite line (Cam3.II R539T) than in the drug-treated isogenic sensitive strain (Cam3.II rev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage.

Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

“…Pre-treatment with E64d and co-incubation with increasing concentrations of OZ03 inhibited OZ727 binding to food vacuole proteins (Figure S2), confirming that these targets are important for peroxide activity. These findings are consistent with peroxides initially acting by disrupting parasite haemoglobin digestion 32 , an essential process that occurs within the parasite food vacuole. GO analysis further revealed that peroxide protein targets were involved in numerous essential biological processes of the parasite (Figure 3 and Supplementary Data 3).…”

“…This process results in cleavage of the peroxide bond and generation of carbon-centered radicals 11,16 that alkylate haem [17][18] and proteins [19][20][21][22][23][24][25][26] and induce widespread oxidative stress [27][28][29][30][31] . Death of the parasite likely results from disruption to multiple vital processes, including haemoglobin degradation in the food vacuole 32 . Concerningly, parasites with decreased sensitivity to artemisinins have emerged in the Greater Mekong Subregion 33 , and more recently in eastern India 34 , Africa 35 and Papua New Guinea 36 .…”

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“…The current portfolio contains a number of promising drug candidates like arterolane (OZ277, Figure 13 , 40 ), artefenomel (OZ439, 41 ) and ferroquine ( 30 ) [ 73 , 74 ]. Artefenomel ( 40 ) and arterolane ( 39 ), like the artemisinins, contain a 1,2,4-trioxane ring and has the same mechanism [ 87 ]. The advantages of these drugs are longer half-lives in the body, 4 hours for arterolane [ 87 ] and 23 hours for artefenomel [ 73 , 87 ] in contrast to the 1 hour half-life of artemisinins.…”

“…Artefenomel ( 40 ) and arterolane ( 39 ), like the artemisinins, contain a 1,2,4-trioxane ring and has the same mechanism [ 87 ]. The advantages of these drugs are longer half-lives in the body, 4 hours for arterolane [ 87 ] and 23 hours for artefenomel [ 73 , 87 ] in contrast to the 1 hour half-life of artemisinins. Long time exposure to the drugs is expected to make Kelch13 mutated parasites more sensitive [ 73 ].…”

“…Long time exposure to the drugs is expected to make Kelch13 mutated parasites more sensitive [ 73 ]. Arterolane ( 40 ) is presently used clinically in combination with primaquine in India [ 73 , 87 ]. Other 1,2,4-trioxanes are also tested.…”