title: Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel).
abstract: The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treatedPlasmodium falciparummalaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treatedBabesia divergens, parasites that also proliferate inside erythrocytes but, in contrast toP. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify theP. falciparumproteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins inP. falciparum.
authors.affiliation: Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland; University of Basel, CH-4003 Basel, Switzerland.
authors.forename: Joëlle
authors.initials: J
authors.lastname: Jourdan
authors.affiliation: F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
authors.forename: Hugues
authors.initials: H
authors.lastname: Matile
authors.affiliation: Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland; University of Basel, CH-4003 Basel, Switzerland.
authors.forename: Ellen
authors.initials: E
authors.lastname: Reift
authors.affiliation: Imaging Core Facility, Biozentrum, University of Basel , CH-4003 Basel, Switzerland.
authors.forename: Oliver
authors.initials: O
authors.lastname: Biehlmaier
authors.affiliation: College of Pharmacy, University of Nebraska Medical Center , 986025 Nebraska Medical Center, Omaha, Nebraska 68198, United States.
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Dong
authors.affiliation: College of Pharmacy, University of Nebraska Medical Center , 986025 Nebraska Medical Center, Omaha, Nebraska 68198, United States.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Wang
authors.affiliation: Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland; University of Basel, CH-4003 Basel, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Mäser
authors.affiliation: College of Pharmacy, University of Nebraska Medical Center , 986025 Nebraska Medical Center, Omaha, Nebraska 68198, United States.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Vennerstrom
authors.affiliation: Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland; University of Basel, CH-4003 Basel, Switzerland.
authors.forename: Sergio
authors.initials: S
authors.lastname: Wittlin
keywords: Plasmodium falciparum; alkylation; artemisinin; immunofluorescence; monoclonal antibody; ozonide
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