A class of valuable (pro-)activity-based protein profiling probes: application to the redox-active antiplasmodial agent, plasmodione

International audience; Plasmodione (PD) is a potent antimalarial redox-active drug acting at low nM range concentrations on different malaria parasite stages. In this study, in order to determine the precise PD protein interactome in parasites, we developed a class of (pro-)activity-based protein profiling probes (ABPP) as precursors of photoreactive benzophenonelike probes based on the skeleton of PD metabolites (PDO) generated in a cascade of redox reactions. Under UV-photoirradiation, we clearly demonstrate that benzylic oxidation of 3-benzylmenadione 11 produces the 3-benzoylmenadione probe 7, allowing investigation of the proof-of-concept of the ABPP strategy with 3-benzoylmenadiones 7-10. The synthesized 3-benzoylmenadiones, probe 7 with an alkyne group or probe 9 with-NO2 in para position of the benzoyl chain, were found to be the most efficient photoreactive and clickable probes. In the presence of various H-donor partners, the UV-irradiation of the photo-reactive ABPP probes generates different adducts, the expected 'benzophenone-like' adducts (pathway 1) in addition to 'benzoxanthone' adducts (via two other pathways, 2 and 3). Using both human and Plasmodium falciparum glutathione reductases three protein ligand binding sites were identified following photolabeling with probes 7 or 9. The photoreduction of 3-benzoylmenadiones (PDO and probe 9) promoting the formation of both the corresponding benzoxanthone and the derived enone could be replaced by the glutathione reductase-catalyzed reduction step. In particular, the electrophilic character of the benzoxanthone was evidenced by its ability to alkylate heme, as a relevant event supporting the antimalarial mode of action of PD. This work provides a proof-of-principle that (pro-)ABPP probes can generate benzophenonelike metabolites enabling optimized activity-based protein profiling conditions that will be instrumental to analyse the interactome of early-lead antiplasmodial 3-benzylmenadiones displaying an original and innovative mode of action.