Stochastic Protein Alkylation by Antimalarial Peroxides

Antimalarial peroxides such as the phytochemical artemisinin
or
the synthetic ozonides arterolane and artefenomel undergo reductive
cleavage of the pharmacophoric peroxide bond by ferrous heme, released
by parasite hemoglobin digestion. The generated carbon-centered radicals
alkylate heme in an intramolecular reaction and proteins in an intermolecular
reaction. Here, we determine the proteinaceous alkylation signatures
of artemisinin and synthetic ozonides in Plasmodium falciparum using alkyne click chemistry probes to identify target proteins
by affinity purification and mass spectrometry-based proteomics. Using
stringent controls and purification procedures, we identified 25 P. falciparum proteins that were alkylated by the antimalarial
peroxides in a peroxide-dependent manner, but the alkylation patterns
were more random than we had anticipated. Moreover, there was little
overlap in the alkylation signatures identified in this work and those
disclosed in previous studies. Our findings suggest that alkylation
of parasite proteins by antimalarial peroxides is likely to be a nonspecific,
stochastic process.