Profiling the Antimalarial Mechanism of Artemisinin by Identifying Crucial Target Proteins

Artemisinin-resistantPlasmodium falciparumKelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation

“…Our results also indicate that some of the AAS generated by the TrK13-heme complex are likely to be soluble, as evidenced by release experiments performed with the TrK13-WT, which suggests that limited proteotoxicity of those AAS directed at TrK13 could occur. This is in line with two reports that did not identify PfKelch13 as alkylation target of artesunate or the alkyne-labeled ART analog AP1 coupled to biotin 6,9 . Nonetheless, the existence of putative TrK13-AAS adducts in our in vitro system should be carefully investigated by appropriate methods.…”

“…P. falciparum 3D7 parasites were grown in human erythrocytes supplemented with 2 mM L-glutamine, 50 mg/l hypoxanthine, 25 mM HEPES, 0.21% NaHCO 3 , 0.1 mg/l penicillin-streptomycin, and 0.5% w/v Albumax II (Invitrogen) under 5% CO 2 and 5% O 2 , at 37°C. Approximately 2 × 10 9 3D7 parasites were isolated by saponin treatment from asynchronized culture (4 flasks of 25 mL culture at 4% parasitemia) and frozen immediately at -80°C as dry pellets (total parasite pellet volume was approximately 100 µl). Total parasite proteins were solubilized with RIPA buffer as follows: 1 volume of dry parasite pellet was resuspended in 10 volumes of ice-cold RIPA buffer complemented with 1X protease inhibitor (cOmplete, Roche).…”

“…These target the C-C, C-H, and other bonds in parasite proteins/lipids, and the resulting alkylation leads to extensive molecular damage and parasite death [3][4][5][6][7]8 . The alkylation of protein targets by ART radicals appears to be rather random, as two different click-chemistry studies using ART probes found little overlap in the set of alkylated parasite proteins 6,9 . Other anti-parasitic mechanisms include inhibition of β-hematin crystallization and heme detoxification by heme-drug adducts and, paradoxically, of endocytosis-mediated uptake of hemoglobin, the source of ART activator 10,11 .…”

Access to Artemisinin–Triazole Antimalarials via Organo-Click Reaction: High In Vitro/In Vivo Activity against Multi-Drug-Resistant Malaria Parasites

“…This resulted in the discovery of compounds of enhanced in vitro antimalarial potency and in vivo efficacy compared to ART. These observations align with the fact that changes in the skeleton and implantation of chemical motifs surrounding the trioxane warhead can modulate the pharmacological activity of ARTs. − More specifically, these structural changes in the endoperoxide drugs can directly impact the speed of heme-mediated peroxide activation and, subsequently, in the protein alkylation profile. − …”

Characterization of antimalarial activity of artemisinin-based hybrid drugs

“…As a reducing agent of peroxide, Fe(II)-heme plays a key role by leading to the formation of a C-centered radical able to alkylate heme and proteins [16]. It is this activation pathway that has been shown to be responsible for the antiplasmodial activity of peroxide-based antimalarials [16][17][18][19]. Despite some subtle differences in the peroxide homolysis products among the ART-derived drugs used clinically [15], the elimination half-life (t 1/2 ) of all these drugs in the plasma is short and considered to be the bottleneck [12,20].…”