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-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins 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 in P. falciparum.

Rearrangements of organic peroxides and related processes

“…Four peaks at m / z 782.3 were detected which were assigned to the four possible regioisomers of alkylated heme adduct 648 as reported for heme–artemisinin adducts [ 558 ]. Later, in an initial study dealing with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of synthetic peroxides it was shown that the artemisinins alkylate proteins in P. falciparum [ 559 ].…”

Increasing the Strength and Production of Artemisinin and Its Derivatives

“…It has been proposed that, like artemisinin, the antimalarial synthetic peroxides also undergo reductive cleavage by the ferrous heme of the catabolized hemoglobin [ 113 ]. This peroxide reduction resulted in active carbon-centered radicals that alkylate heme and the parasite proteins [ 113 ].…”

In vitro activity of anti-malarial ozonides against an artemisinin-resistant isolate

“…Since the structural analogies between artemisinins and ozonides (Fig. 1 ) suggest that they share similar modes of action, and thus some level of cross resistance [ 9 , 10 , 51 , 52 ], the per cent survival of an artemisinin-resistant clinical isolate (Cam3.I R539T ) treated with DHA, OZ277, OZ439, and four additional next-generation ozonides (Fig. 1 ) using the RSA 0–3h as described by Witkowski et al [ 48 , 49 ] was evaluated.…”