Stochastic Protein Alkylation by Antimalarial Peroxides

Abstract

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.

Artemisinin inspired synthetic endoperoxide drug candidates: Design, synthesis, and mechanism of action studies

“…Another downside is that there wasn't enough correlation in the alkylated proteins identified between all findings, 46,[67][68][69] which can be explained by the differences in the conditions used in each study. However, these results can also suggest that activated endoperoxides unsystematically and nonspecifically alkylate parasite proteins, thus inhibiting essential proteins involved in key parasite pathways, in a multiple target approach.…”

“…In three studies recently undertaken, alkylated P. falciparum proteins were identified using activity-based probes of artemisinin (12-16, Figure 3). 46,[67][68][69] Infected P. falciparum erythrocytes were treated with endoperoxide probes. Following incubation, if probes were active against the parasite, they would promote activation and lead to parasite proteins' covalent binding.…”

“…The probes consisted of an antimalarially active endoperoxide tagged with biotin or a fluorescent label, via a click reaction, and the alkylated proteins could be identified by mass spectrometry techniques (LC-MS/MS) or processed in 1D-Gel analysis. 46,[67][68][69] Wang et al 46 used a non-optimized artemisinin-alkyne activity-based (12, Figure 3) protein-profiling probe associated with 124 artemisinin-alkylated proteins through click-chemistry procedures. The addition of free-iron chelator deferoxamine, in the presence of hemin or heme, was found to decrease the alkylation of a few protein targets slightly, but not all of them.…”

Plasmodium falciparum resistance to ACTs: Emergence, mechanisms, and outlook

“…Early studies detected proteins that are labeled by radioactive endoperoxides ( Asawamahasakda et al, 1994 ) and characterized one of these proteins – the translationally controlled tumor protein PfTCTP ( Bhisutthibhan and Meshnick, 2001 ; Li et al, 2016 ). However, recent proteomic studies indicate that the ART alkylation activity is promiscuous, involving hundreds of targets across many essential biological processes ( Ismail et al, 2016a , Ismail et al, 2016b ; Jourdan et al, 2019 ; Wang et al, 2015a ). Given that this experimental procedure cannot identify non-covalent targets, the ART target spectrum may be much broader.…”

Techniques and Strategies for Potential Protein Target Discovery and Active Pharmaceutical Molecule Screening in a Pandemic

“…For example, Zhang et al 87 achieved immunofluorescence colocalization with the natural inhibitor AKT through the click reaction between the probe and the alkynyl CY5, further illustrating the anti-inflammatory mechanism of Swertiamarin, the component of honeysuckle. Jourdan et al 88 found that antimalarial drugs invading pathogens are a random alkylation process, and using click chemistry and other technologies found that 25 proteins changed. However, the disadvantage of using this method is that the introduction of biorthogonal groups such as alkynyl groups on small molecules through organic synthesis will lead to a decrease in the activity of the compound.…”