title: Haemoglobin degradation underpins the sensitivity of early ring stage Plasmodium falciparum to artemisinins.
abstract: Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Stanley C
authors.initials: SC
authors.lastname: Xie
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Con
authors.initials: C
authors.lastname: Dogovski
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia Advanced Microscopy Facility, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Eric
authors.initials: E
authors.lastname: Hanssen
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Melbourne, Victoria 3010, Australia.
authors.forename: Francis
authors.initials: F
authors.lastname: Chiu
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Tuo
authors.initials: T
authors.lastname: Yang
authors.affiliation: Department of Microbiology, University of Valle, 13 #100-00, Cali, Valle del Cauca, Colombia Department of Biomedical Sciences, Santiago de Cali University, 25, Cali, Valle del Cauca, Colombia.
authors.forename: Maria P
authors.initials: MP
authors.lastname: Crespo
authors.affiliation: Walter+Eliza Hall Institute, Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3052, Australia.
authors.forename: Che
authors.initials: C
authors.lastname: Stafford
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Steven
authors.initials: S
authors.lastname: Batinovic
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Silvia
authors.initials: S
authors.lastname: Teguh
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Melbourne, Victoria 3010, Australia.
authors.forename: Susan
authors.initials: S
authors.lastname: Charman
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Nectarios
authors.initials: N
authors.lastname: Klonis
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia LTilley@unimelb.edu.au.
authors.forename: Leann
authors.initials: L
authors.lastname: Tilley
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004351:Drug Resistance; D004357:Drug Synergism; D006454:Hemoglobins; D006801:Humans; D007928:Lethal Dose 50; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D016778:Malaria, Falciparum / Q000188:drug therapy; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology; D059748:Proteolysis; D015800:Protozoan Proteins / Q000378:metabolism
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D015853:Cysteine Proteinase Inhibitors; D006454:Hemoglobins; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C099607:falcipain; D007930:Leucine; C108192:aloxistatin
keywords: Artemisinin; Falcipains; Haemoglobin degradation; Malaria; Plasmodium; Resistance