Identification of compounds active against quiescent artemisinin-resistant Plasmodium falciparum parasites via the quiescent-stage survival assay (QSA)

Abstract

Background Quiescence is an unconventional mechanism of Plasmodium survival, mediating artemisinin resistance. This phenomenon increases the risk of clinical failures following artemisinin-based combination therapies (ACTs) by slowing parasite clearance and allowing the selection of parasites resistant to partner drugs. Objectives To thwart this multiresistance, the quiescent state of artemisinin-resistant parasites must be taken into consideration from the very early stages of the drug discovery process. Methods We designed a novel phenotypic assay we have named the quiescent-stage survival assay (QSA) to assess the antiplasmodial activity of drugs on quiescent parasites. This assay was first validated on quiescent forms from different artemisinin-resistant parasite lines (laboratory strain and field isolates), using two reference drugs with different mechanisms of action: chloroquine and atovaquone. Furthermore, the efficacies of different partner drugs of artemisinins used in ACTs were investigated against both laboratory strains and field isolates from Cambodia. Results Our results highlight that because of the mechanism of quiescence and the respective pharmacological targets of drugs, drug efficacies on artemisinin-resistant parasites may be different between quiescent parasites and their proliferating forms. Conclusions These data confirm the high relevance of adding the chemosensitivity evaluation of quiescent parasites by the specific in vitro QSA to the antiplasmodial drug development process in the current worrisome context of artemisinin resistance.

2-Phenoxy-3-Trichloromethylquinoxalines Are Antiplasmodial Derivatives with Activity against the Apicoplast of Plasmodium falciparum

“…However, despite ART-resistant genotype of F32-ART5, EC 50 values of ART were also similar for both strains (ranging from 11 to 19 nM, Mann–Whitney test p-value = 0.267) ( Table 4 ), due to the specific P. falciparum quiescence-based mechanism of ART-resistance leading to parasite cell cycle arrest during drug exposure. Therefore, evaluation of ( 3i ) with specific assays of artemisinin-resistance [ 33 , 34 , 35 ] was conducted and evidenced that this compound was active against the artemisinin-resistant parasites at the proliferative state but not at the quiescent state (see Supplementary Materials Figure S49 ).…”

“…Chemosensitivity evaluation of ART-resistant parasites at the quiescent stage was performed on the strain F32-ART5 thanks to the quiescent-stage survival assay (QSA) [ 35 ]. D-sorbitol synchronized ring-stages parasites at 3% parasitemia (2% hematocrit) were first exposed to 700 nM of dihydroartemisinin (DHA) for 6 h to induce quiescence of artemisinin-resistant parasites.…”

“…In each experiment, results obtained with the condition “DHA 6 h/DHA 48 h” were compared to those obtained with the condition “DHA 6 h/(DHA + drug to be tested) 48 h” in order to determine the delay in recrudescence time. This delay is representative of the capacity of the tested compound to be active on quiescent parasites and it is assumed that a 6-day threshold is necessary to classify a compound as active on quiescent parasites [ 35 ].…”

Novel molecule combinations and corresponding hybrids targeting artemisinin-resistant Plasmodium falciparum parasites

“…10,13,14 Interestingly, the antimalarial drug atovaquone (ATQ, Table 1), which targets the bc1 complex of mitochondrial electron transport chain, 15 was reported to kill dihydroartemisinin-induced quiescent parasites. 16,17 Similarly, triclosan (TCS) and haloxyfop, which inhibit the FabI and acetyl-CoA carboxylase of the Plasmodium FAS-II pathway respectively, delayed the recrudescence of quiescent parasites after dihydroartemisinin treatment. 13 Simultaneously targeting the mitochondrion with ATQ and the apicoplast with TCS would thus be of valuable interest to face the issue of ART-resistance.…”

“…Finally, the antimalarial drug mefloquine (MQ) was picked out as a relevant compound for drug combinations studies because it is one of the only two partner drugs used in ACTs to be reported as active on quiescent ART-resistant parasites. [17] We report here the evaluation of different combinations of two of these compounds, each targeting one essential pathway for quiescence survival: ATQ + TCS, ATQ + MQ, GW844520 + TCS. This evaluation was performed first on proliferating parasites then in an artemisinin resistance context on dihydroartemisinin (DHA)induced quiescent state, thanks to two specific tests, the recrudescence assay, 19 and the Quiescent-stage Survival Assay (QSA) for the best combination.…”