Direct evidence for the atovaquone action on the Plasmodium cytochrome bc 1 complex

130

e High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum. In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-D-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs. Malaria remains a major global health problem, and there is a pressing need to discover and develop new antimalarials. Traditional antimalarial drugs have been severely undermined by the emergence of drug-resistant strains and current treatments rely heavily on artemisinin-based combination therapies. Alarmingly, artemisinin resistance has arisen in Southeast Asia during the last decade, highlighting the urgent need for new antimalarials (1). Extensive efforts to produce a malaria vaccine have met limited success and a pipeline of new antimalarial drugs will be required to mitigate extensive morbidity and mortality from malaria for the foreseeable future (2).High-throughput phenotypic screening of chemical libraries against the malaria parasite, Plasmodium falciparum, has provided a major step forward in the discovery of novel antimalarial compounds. Almost 30,000 compounds that selectively inhibit growth of cultured P. falciparum asexual red blood cell (RBC) stages have been identified in these screens, providing excellent starting points for the discovery of new antimalarial drugs (3-5). A prioritized collection of these "hit" compounds, known as the Malaria Box, has been assembled by the Medicines for Malaria Venture (MMV) and provided free to the research community to facilitate this drug development pipeline (6).A key limitation to the further optimization of many of these compounds is the lack of information on their mode of action. While not essential for registration, information on the mode of action of inhibitors discovered in phenotypic screens will significantly accelerate drug development by allowing structure-based drug design, monitoring of activity, toxicity and resistance, and facilitation of rational clinical usage in combination with other medicines. Furthermore, in...

“…fected by mutations in the target proteins that confer resistance to atovaquone or DSM265 (51,52). Identification of a specific inhibitor of isoprenoid precursor biosynthesis.…”

Section: Resultsmentioning

confidence: 99%

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

Creek

Chua

Cobbold

et al. 2016

130

“…Various single nucleotide polymorphisms (SNPs) in the quinine binding site of the Plasmodium species cytochrome b (cytb) gene have been implicated in conferring resistance to ATQ (6). While M133I and L271V have been implicated in murine malarial models (7,8), and L144S, K272R, and V284F have been demonstrated in cultures exposed to high concentrations of ATQ (9), SNPs in position 268 (Y268C, Y268S, and Y268N) have been associated with clinical failure (10). ATQ resistance also appears to be associated with the delayed recrudescence of resistant parasites Ն3 weeks after initial clearance of parasitemia by ATQ-PG therapy (11).…”

mentioning

confidence: 99%

Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

Saunders

Chaorattanakawee

Gosi

et al. 2016

Our recent report of dihydroartemisinin-piperaquine failure to treat Plasmodium falciparum infections in Cambodia adds new urgency to the search for alternative treatments. Despite dihydroartemisinin-piperaquine failure, and higher piperaquine 50% inhibitory concentrations (IC 50 s) following reanalysis than those previously reported, P. falciparum remained sensitive to atovaquone (ATQ) in vitro. There were no point mutations in the P. falciparum cytochrome b ATQ resistance gene. Mefloquine, artemisinin, chloroquine, and quinine IC 50 s remained comparable to those from other recent reports. Atovaquone-proguanil may be a useful stopgap but remains susceptible to developing resistance when used as blood-stage therapy.

“…3, showing the transmembrane arrangement of the apoprotein, while a similar repeated incomplete treatment (RIcT) but with a higher, therapeutic dose of the drug led to resistance mutations mostly in the Qo2 domain. The sites correspond to the degree of resistance conferred by the mutations; the level of atovaquone resistance associated with mutations in the Qo1 domain has been shown to be at least 20 times lower than that associated with mutations in the Qo2 domain in P. berghei (8). Atovaquone resistance mutations in both Qo1 (M133I) and Qo2 (Y268S) have been reported for the human malaria parasite Plasmodium falciparum.…”

Section: Discussionmentioning

confidence: 99%

“…The relationship between mutations in the Qo1 and Qo2 domains of the cytochrome b protein and the degree of resistance to atovaquone has been reported (4,8). A structural interaction between atovaquone and the cytochrome b protein has also been proposed (9).…”

Section: Dose-dependent Selection Of Atovaquone Resistance Antimicrobmentioning

confidence: 99%

“…Almost all mutations underlying atovaquone resistance of P. berghei resulting from RIcT selection were found to be in the Qo2 (quinone binding 2) domain of the cytochrome b gene (Y268C/N/S, L271V, and K272R), associated with relatively high degrees of resistance in vivo as well as in vitro (7,8). Previous attempts to isolate atovaquone-resistant mutants of P. berghei by the serial technique (ST) method, which involves serial passages of parasites in mice treated with gradually increasing drug doses (5), on the other hand, resulted in atovaquone resistance associated with mutations that are predominantly in the transmembrane (V284F) or in the Qo1 (M133I and L144S) domain of the cytochrome b gene.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Within-Host Selection of Drug Resistance in a Mouse Model Reveals Dose-Dependent Selection of Atovaquone Resistance Mutations

Nuralitha

Murdiyarso

Siregar

et al. 2017

Self Cite

The evolutionary selection of malaria parasites within an individual host plays a critical role in the emergence of drug resistance. We have compared the selection of atovaquone resistance mutants in mouse models reflecting two different causes of failure of malaria treatment, an inadequate subtherapeutic dose and an incomplete therapeutic dose. The two models are based on cycles of insufficient treatment of Plasmodium berghei-infected mice: repeated inadequate treatment associated with a subtherapeutic dose (RIaT) (0.1 mg kg Ϫ1 of body weight) and repeated incomplete treatment with a therapeutic dose (RIcT) (14.4 mg kg Ϫ1 of body weight). The number of treatment cycles for the development of a stable resistance phenotype during RIaT was 2.00 Ϯ 0.00 cycles (n ϭ 9), which is not statistically different from that during RIcT (2.57 Ϯ 0.85 cycles; combined n ϭ 14; P ϭ 0.0591). All mutations underlying atovaquone resistance selected by RIaT (M133I, T142N, and L144S) were found to be in the Qo1 (quinone binding 1) domain of the mitochondrial cytochrome b gene, in contrast to those selected by RIcT (Y268N/C, L271V, K272R, and V284F) in the Qo2 domain or its neighboring sixth transmembrane region. Exposure of mixed populations of resistant parasites from RIaT to the higher therapeutic dose of RIcT revealed further insights into the dynamics of within-host selection of resistance to antimalarial drugs. These results suggest that both inadequate subtherapeutic doses and incomplete therapeutic doses in malaria treatment pose similar threats to the emergence of drug resistance. RIcT and RIaT could be developed as useful tools to predict the potential emergence of resistance to newly introduced and less-understood antimalarials.KEYWORDS dose-dependent selection, mouse malaria model, repeated inadequate treatment, repeated incomplete treatment, within-host selection of atovaquone resistance T he evolutionary selection of malaria parasites within an individual host plays a critical role in the emergence of antimalarial drug resistance, a major problem in malaria control. The study of within-host selection of drug resistance benefits from animal models of malaria infection, as it allows pharmacological manipulations in vivo.We recently reported such a model, based on cycles of repeated incomplete treatment (RIcT) of Plasmodium berghei-infected mice with a constant therapeutic dose of an antimalarial drug, which mimics treatment failure in the human field situation (1). We showed that under these conditions, stable resistance of P. berghei to the antimalarial atovaquone developed rapidly, after only 2.5 cycles of treatment.