*3.3. Protein Damage*

Proteins are vulnerable to oxidation by ROS, a phenomenon generally referred to as protein carbonylation. Protein carbonyls are reactive aldehydes and ketone adducts formed through the α-amidation pathway, the formation of protein–protein cross-linked derivatives, the oxidative cleavage of glutamyl residues, and cell membrane damage by lipid oxidation products [28]. Protein carbonyls have been used as biomarkers of oxidative stress due to their relative stability and early formation [29]. ROS generate cytotoxic protein carbonyls in two main and irreversible ways:


These post-translational modifications due to the oxidization of amino acid side chains, which results in aldehyde, ketone, and lactam formation, can also lead to damage to proteins [30,31].

The endoperoxides alkylate numerous vital proteins, including enzymes in the parasite, particularly the cysteine residue of cysteine proteases, which play a vast role in *P. falciparum*, ranging from hemoglobin (Hb) uptake and digestion to aiding red blood cell rupture [8,32,33]. Therefore, their alteration, leading to the inhibition of their enzymatic properties, can cause severe setbacks for parasite survival, considering the huge dependence of the parasite on Hb digestion products. Endoperoxides also disrupt the activities of sarcoplasmic–endoplasmic reticulum Ca2+-ATPase- (SERCA-) type protein, encoded by the *pfatpase6* gene, presumably from the ROS that they generate [34]. More recently, approximately 124 proteins were identified as covalent binding targets of artemisinins [35].

**Figure 4.** Typical carbonylation of proteins based on a metal-catalyzed oxidation (MCO) attack involving a transition metal (Fe2+) for hydroxyl radical generation.

#### *3.4. Nucleic Acid Damage*

The molecular integrity of DNA and other genetic material is necessary for the continued existence and survival of all living organisms, including *Plasmodium*. ROS cause structural damage to DNA by attacking mainly one of its bases, guanine, due to its lower oxidation potential [36,37] (Figure 5). Reports have revealed that artesunate can cause double-stranded breaks of plasmodial DNA in less than one hour and that it is linked to an increase in ROS generation [38].

**Figure 5.** DNA base damage by ROS at the purine base guanine. The C8-OH adduct 7-hydro8-oxo-2'-deoxyguanosine is used as a marker of oxidative stress. A similar reaction occurs with adenine (8-oxoAde and Fapy-Ade). FapyGua: formamidopyrimidine, 8-oxoGua: 8-oxoguanine [36,39].

> Disruption of the conformation of biomolecules by artemisinins (endoperoxides) or other ROS-generating antimalarials kills parasites. Although they have a similar activation process generating carbon- and oxygen-centered radicals, the efficacy of endoperoxides varies, which can be explained, in part, by their different pharmacological properties, notably the stage of the parasite erythrocyte cycle, the nature of their target, and their location in the parasite, i.e., trioxolanes vs. artemisinin and its derivatives [26,40].
