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

View ORCID ProfileCarlo Giannangelo, Ghizal Siddiqui, Amanda De Paoli, View ORCID ProfileBethany M. Anderson, Laura E. Edgington-Mitchell, View ORCID ProfileSusan A. Charman, View ORCID ProfileDarren J. Creek
doi: https://doi.org/10.1101/2020.03.23.003376
Carlo Giannangelo
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
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  • ORCID record for Carlo Giannangelo
Ghizal Siddiqui
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
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Amanda De Paoli
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
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Bethany M. Anderson
2Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia
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Laura E. Edgington-Mitchell
2Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia
4Department of Maxillofacial Surgery, College of Dentistry, New York University, New York, New York, USA
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Susan A. Charman
5Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
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Darren J. Creek
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria Australia
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  • For correspondence: Darren.creek@monash.edu
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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. The association between ozonide activity and Hb catabolism was also confirmed in a K13-mutant artemisinin resistant parasite line. 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.

Author Summary The ozonides are a novel class of fully synthetic antimalarial drugs with potent activity against all parasite species that cause malaria, including the deadliest, Plasmodium falciparum. With the emergence of resistance to current frontline artemisinin-based antimalarials, new drugs are urgently needed and a clear understanding of their mechanism of action is essential so that they can be optimally deployed in the field. Here, we studied the biochemical effects of two ozonides, OZ277 (marketed in India in combination with piperaquine) and OZ439 (in Phase IIb clinical trials) in P. falciparum parasites using an untargeted multi-omics approach consisting of proteomics, peptidomics and time-dependent metabolomics, along with activity-based protease profiling. We found that the ozonides initially disrupt haemoglobin metabolism and that they likely engage the parasite proteostatic stress response. Furthermore, when the duration of ozonide exposure was extended beyond 3 hours to reflect clinically-relevant exposure periods, additional parasite biochemical pathways were perturbed. This comprehensive analysis provides new insight into the antimalarial mode of action of ozonides and provides new opportunities for interventions to enhance their antimalarial efficacy.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted March 23, 2020.
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System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion
Carlo Giannangelo, Ghizal Siddiqui, Amanda De Paoli, Bethany M. Anderson, Laura E. Edgington-Mitchell, Susan A. Charman, Darren J. Creek
bioRxiv 2020.03.23.003376; doi: https://doi.org/10.1101/2020.03.23.003376
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System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion
Carlo Giannangelo, Ghizal Siddiqui, Amanda De Paoli, Bethany M. Anderson, Laura E. Edgington-Mitchell, Susan A. Charman, Darren J. Creek
bioRxiv 2020.03.23.003376; doi: https://doi.org/10.1101/2020.03.23.003376

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