Antimalarial Atovaquone Research Articles

Computational exploration of compounds in Xylocarpus granatum as a potential inhibitor of Plasmodium berghei using docking, molecular dynamics, and DFT studies

Malaria remains a global health concern, with the emergence of resistance to the antimalarial drug atovaquone through cytochrome b (cyt b) being well-documented. This study was prompted by the presence of this mutation in cyt b to enable new drug candidates capable of overcoming drug resistance. Our objective was to identify potential drug candidates from compounds of Xylocarpus granatum by computationally assessing their interactions with Plasmodium berghei cyt b. Using computational methods, we modeled cyt b (GenBank: AF146076.1), identified the binding cavity, and analyzed the Ramachandran plot against cyt b. Additionally, we conducted drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies, along with density functional theory (DFT) analysis of the compounds. Molecular docking and molecular dynamics simulation (MDS) were used to evaluate the binding energy and stability of the cyt b-ligand complex. Notably, our investigation highlighted kaempferol as a promising compound due to its high binding energy of 7.67 kcal/mol among all X. granatum compounds, coupled with favorable pharmacological properties (ADMET) and antiprotozoal properties at Pa 0.345 > Pi 0.009 (PASS value). DFT analysis showed that kaempferol has an energy gap of 4.514 eV. MDS indicated that all tested ligands caused changes in bonding and affected the structural conformation of cyt b, as observed before MDS (0 ns) and after MDS (100 ns). The most notable differences were observed in the types of hydrogen bonds between 0 and 100 ns. Nevertheles, MDS results from a 100 ns simulation revealed consistent behavior for kaempferol across various parameters including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), molecular mechanics-Poisson Boltzmann surface area (MM-PBSA), and hydrogen bonds. The cyt b-kaempferol complex demonstrated favorable energy stability, as supported by the internal energy distribution values observed in principal component analysis (PCA), which closely resembled those of the atovaquone control. Additionally, trajectory stability analysis indicated structural stability, with a cumulative eigenvalue of 24.7 %. Dynamic cross-correlation matrix (DCCM) analysis revealed a positive correlation among catalytic cytochrome residues within the amino acid residues range 119–268. The results of our research indicate that the structure of kaempferol holds promise as a potential candidate against Plasmodium.

Plasmodium falciparum Mitochondrial Complex III, the Target of Atovaquone, Is Essential for Progression to the Transmissible Sexual Stages.

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

MalariaFlow: A comprehensive deep learning platform for multistage phenotypic antimalarial drug discovery

Malaria remains a significant global health challenge due to the growing drug resistance of Plasmodium parasites and the failure to block transmission within human host. While machine learning (ML) and deep learning (DL) methods have shown promise in accelerating antimalarial drug discovery, the performance of deep learning models based on molecular graph and other co-representation approaches warrants further exploration. Current research has overlooked mutant strains of the malaria parasite with varying degrees of sensitivity or resistance, and has not covered the prediction of inhibitory activities across the three major life cycle stages (liver, asexual blood, and gametocyte) within the human host, which is crucial for both treatment and transmission blocking. In this study, we manually curated a benchmark antimalarial activity dataset comprising 407,404 unique compounds and 410,654 bioactivity data points across ten Plasmodium phenotypes and three stages. The performance was systematically compared among two fingerprint-based ML models (RF::Morgan and XGBoost:Morgan), four graph-based DL models (GCN, GAT, MPNN, and Attentive FP), and three co-representations DL models (FP-GNN, HiGNN, and FG-BERT), which reveal that: 1) The FP-GNN model achieved the best predictive performance, outperforming the other methods in distinguishing active and inactive compounds across balanced, more positive, and more negative datasets, with an overall AUROC of 0.900; 2) Fingerprint-based ML models outperformed graph-based DL models on large datasets (>1000 compounds), but the three co-representations DL models were able to incorporate domain-specific chemical knowledge to bridge this gap, achieving better predictive performance. These findings provide valuable guidance for selecting appropriate ML and DL methods for antimalarial activity prediction tasks. The interpretability analysis of the FP-GNN model revealed its ability to accurately capture the key structural features responsible for the liver- and blood-stage activities of the known antimalarial drug atovaquone. Finally, we developed a web server, MalariaFlow, incorporating these high-quality models for antimalarial activity prediction, virtual screening, and similarity search, successfully predicting novel triple-stage antimalarial hits validated through experimental testing, demonstrating its effectiveness and value in discovering potential multistage antimalarial drug candidates.

Tarsal exposure to atovaquone inhibits chikungunya virus transmission by Aedes aegypti mosquitoes, but not the transmission of Zika virus

The antimalarial drug atovaquone was recently reported to inhibit the in vitro replication of different arboviruses, including chikungunya virus (CHIKV) and Zika virus (ZIKV). Furthermore, atovaquone was shown to block Plasmodium parasite transmission by Anopheles mosquitoes when the mosquitoes were exposed to low concentrations on treated surfaces (i.e. tarsal exposure). Therefore, we evaluated the anti-CHIKV and -ZIKV effects of atovaquone via tarsal exposure in Aedes aegypti mosquitoes. We first confirmed that atovaquone exerted a dose-dependent antiviral effect on CHIKV and ZIKV replication in mosquito-derived cells. The modest antiviral effect could be rescued by adding exogenous uridine. Next, we assessed the effect of tarsal exposure to atovaquone on the fitness of Ae. aegypti. Concentrations up to 100 μmol/m2 did not affect the fecundity and egg-hatching rate. No significant effect on mosquito survival was observed when mosquitoes were exposed to concentrations up to 25 μmol/m2. To evaluate the antiviral effect of atovaquone against CHIKV, we exposed female mosquitoes to 100 μmol/m2 atovaquone for 1h, after which the mosquitoes were immediately infected with CHIKV or ZIKV via bloodmeal. Atovaquone did not significantly reduce ZIKV or CHIKV infection in Ae. aegypti, but successfully blocked the transmission of CHIKV in saliva. Tarsal exposure to antiviral drugs could therefore be a potential new strategy to reduce virus transmission by mosquitoes.

Quantification of Plasmodium falciparum HRP-2 as an alternative method to [3H]hypoxanthine incorporation to measure the parasite reduction ratio in vitro

In the absence of a highly efficacious vaccine, chemotherapy remains the cornerstone to control malaria morbidity and mortality. The threat of the emergence of parasites resistant to artemisinin-based combination therapies highlights the need for new antimalarial drugs ideally with superior properties. The killing rate reflects the speed of action of antimalarial drugs that can be measured in vitro through the parasite reduction ratio (PRR) assay to shortlist interesting candidates. As a standard, the in vitro PRR assay is performed by measuring [3H]hypoxanthine incorporation of Plasmodium falciparum. This methodology is restricted to specialized laboratories due to the handling of radioactive material. In this work, we describe the sandwich enzyme-linked immunosorbent assay to detect P. falciparum histidine-rich protein 2 (HRP-2) as an alternative methodology to assess the PRR. We first validated the methodology with established antimalarial drugs (artesunate, chloroquine, pyrimethamine, and atovaquone) by comparing our results to previous results of the [3H]hypoxanthine incorporation readout provided by an expert lab and subsequently assessed the speed of action of four new antimalarial candidates (compound 22, chlorotonil A, boromycin, and ivermectin). The HRP-2 PRR assay obtained comparable results to the hypoxanthine incorporation readout in terms of parasite growth rate over time, lag phase, and parasite clearance time. In addition, parasite growth following drug exposure was quantified after 7, 14, 21, and 28 days of recovery time. In conclusion, the PRR assay based on HRP-2 is similar to [3H]hypoxanthine in determining a drug´s parasite-killing rate and can be widely used in all research laboratories.

Atovaquone exerts its anticancer effect by inhibiting Na+/K+-ATPase ion transport in canine cancer cells.

New anticancer drugs are being developed to avoid the toxicity and chemoresistance of the currently available drugs. The Food and Drug Administration-approved anti-malarial drug atovaquone is known to act as a selective oxidative phosphorylation inhibitor in the mitochondria by competing with CO Q10 (mitochondrial complex II and III). This study aimed to investigate the effect of atovaquone by examining the Na+/K+-ATPase (NKA) activity in various canine cell lines. Canine cell lines were treated with various concentrations (2.5, 5, 10, 15, and 20 μM) of atovaquone for 24, 48, and 72 h. Human cell lines were used as a control to validate the canine cancer cell lines. The activities of the drugs against the cancer cell lines were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromideassay. The cell metabolic activity was determined by measuring the activities of the nicotinamide adenine dinucleotide phosphate-dependent cellular oxidoreductase enzymes. The NKA activity was measured using the single-cell patch clamping assay. Atovaquone-induced apoptosis by elevating the concentration of reactive oxygen species (ROS) in the tumor cells, leading to cell death. Treatment of canine cancer cells with N-acetylcysteine (ROS inhibitor) reduced the activity of the drug. Furthermore, atovaquone inhibited more than 45% of the NKA ion current. This study demonstrated effects of atovaquone against canine cancer cell lines. The data may prove beneficial in repurposing the drug as a new anticancer agent in canine clinical trials, which might aid in fighting human cancer.

Drug Self-Delivery Nanocubes Enhance O2 -Economized Photodynamic-Immunotherapy of Triple-Negative Breast Cancer by Downregulating Wnt/β-catenin Signaling.

Although the combination of chemotherapy and immune checkpoint inhibitors (ICIs) can treat triple-negative breast cancer (TNBC), the severe effects of chemotherapy on immune cells significantly reduce the efficacy of the ICIs. Photodynamic therapy (PDT) with high selectivity is an alternative to chemotherapy that can also effectively treat hypoxic TNBC. However, high levels of immunosuppressive cells, and low infiltration of cytotoxic T lymphocytes (CTLs) limit the efficacy of PDT combined with ICIs. This study aims to evaluate the role of drug self-delivery nanocubes (ATO/PpIX-SMN) combined with anti-PD-L1 in TNBC treatment. Anti-malarial atovaquone (ATO) enhances protoporphyrin IX (PpIX)-mediated PDT-induced immunogenic cell death and downregulates tumor Wnt/β-catenin signaling. Furthermore, the nanocubes combined with anti-PD-L1, which synergistically induce maturation of dendritic cells, promote infiltration of CTLs, reduce regulatory T cells, and significantly activate the host immune system, thus treating primary and distal tumors. This work demonstrates that ATO/PpIX-SMN can enhance the response rate of anti-PD-L1 in TNBC treatment via O2 -economized photodynamic-downregulating Wnt/β-catenin signaling.

Siccanin Is a Dual-Target Inhibitor of Plasmodium falciparum Mitochondrial Complex II and Complex III

Plasmodium falciparum contains several mitochondrial electron transport chain (ETC) dehydrogenases shuttling electrons from the respective substrates to the ubiquinone pool, from which electrons are consecutively transferred to complex III, complex IV, and finally to the molecular oxygen. The antimalarial drug atovaquone inhibits complex III and validates this parasite’s ETC as an attractive target for chemotherapy. Among the ETC dehydrogenases from P. falciparum, dihydroorotate dehydrogenase, an essential enzyme used in de novo pyrimidine biosynthesis, and complex III are the two enzymes that have been characterized and validated as drug targets in the blood-stage parasite, while complex II has been shown to be essential for parasite survival in the mosquito stage; therefore, these enzymes and complex II are considered candidate drug targets for blocking parasite transmission. In this study, we identified siccanin as the first (to our knowledge) nanomolar inhibitor of the P. falciparum complex II. Moreover, we demonstrated that siccanin also inhibits complex III in the low-micromolar range. Siccanin did not inhibit the corresponding complexes from mammalian mitochondria even at high concentrations. Siccanin inhibited the growth of P. falciparum with IC50 of 8.4 μM. However, the growth inhibition of the P. falciparum blood stage did not correlate with ETC inhibition, as demonstrated by lack of resistance to siccanin in the yDHODH-3D7 (EC50 = 10.26 μM) and Dd2-ELQ300 strains (EC50 = 18.70 μM), suggesting a third mechanism of action that is unrelated to mitochondrial ETC inhibition. Hence, siccanin has at least a dual mechanism of action, being the first potent and selective inhibitor of P. falciparum complexes II and III over mammalian enzymes and so is a potential candidate for the development of a new class of antimalarial drugs.

Synthesis and Antimalarial Activities of New Hybrid Atokel Molecules.

The currently spreading resistance of the malaria parasite Plasmodium falciparum to artemisinin‐based combination therapies makes an urgent need for new efficient drugs. Aiming to kill artemisinin‐resistant Plasmodium, a series of novel hybrid drugs named Atokels were synthesized and characterized. Atokels are based on an 8‐amino‐ or 8‐hydroxyquinoline entity covalently bound to a 1,4‐naphthoquinone through a polyamine linker. These drugs have been designed to target the parasite mitochondrion by their naphthoquinone moiety reminiscent of the antimalarial drug atovaquone, and to trigger a damaging oxidative stress due to their ability to chelate metal ions in order to generate redox active complexes in situ. The most effective Atokel drug shown a promising antimalarial activity (IC50=622 nm on an artemisinin‐resistant P. falciparum strain) and no cytotoxicity at 50 μm indicating a specific antiplasmodial mode of action.

Combining PEGylated mito-atovaquone with MCT and Krebs cycle redox inhibitors as a potential strategy to abrogate tumor cell proliferation

Glycolytic and mitochondrial oxidative metabolism, which are two major energy sources in tumors, are potential targets in cancer treatment. Metabolic reprogramming from glycolysis to mitochondrial oxidative metabolism and vice versa is an adaptive strategy with which tumor cells obtain energy to survive and thrive under the compromised conditions of glycolysis and mitochondrial respiration. Developing highly potent, nontoxic, and tumor-selective oxidative phosphorylation (OXPHOS) inhibitors may help advance therapeutic targeting of mitochondrial drugs in cancer. The FDA-approved antimalarial drug atovaquone (ATO), a mitochondrial complex III inhibitor, was repurposed in cancer treatment. Here, we developed a new class of PEGylated mitochondria-targeted ATO (Mito-(PEG)n-ATO). Depending on the PEGylation chain length (n), Mito-PEG-ATO analogs inhibit both mitochondrial complex I- and complex III-induced oxygen consumption in human pancreatic (MiaPaCa-2) and brain (U87MG) cancer cells. Mito-PEG5-ATO is one of the most potent antiproliferative mitochondria-targeted compounds (IC50 = 38 nM) in MiaPaCa-2 cells, and is more effective than other inhibitors of OXPHOS in MiaPaCa-2 and U87MG cells. Furthermore, we show that the combined use of the most potent OXPHOS-targeted inhibitors (Mito-PEG5-ATO) and inhibitors of monocarboxylate transporters (MCT-1 and MCT-4), Krebs cycle redox metabolism, or glutaminolysis will synergistically abrogate tumor cell proliferation. Potential clinical benefits of these combinatorial therapies are discussed.

Study of Tissue-Specific Reactive Oxygen Species Formation by Cell Membrane Microarrays for the Characterization of Bioactive Compounds.

The production of reactive oxygen species (ROS) increases considerably in situations of cellular stress, inducing lipid peroxidation and multiple alterations in proteins and nucleic acids. However, sensitivity to oxidative damage varies between organs and tissues depending on the triggering process. Certain drugs used in the treatment of diverse diseases such as malaria have side effects similar to those produced by oxidative damage, although no specific study has been conducted. For this purpose, cell membrane microarrays were developed and the superoxide production evoked by the mitochondrial activity was assayed in the presence of specific inhibitors: rotenone, antimycin A and azide. Once the protocol was set up on cell membrane isolated from rat brain areas, the effect of six antimalarial drugs (atovaquone, quinidine, doxycycline, mefloquine, artemisinin, and tafenoquine) and two essential oils (Rosmarinus officinalis and Origanum majoricum) were evaluated in multiple human samples. The basal activity was different depending on the type of tissue, the liver, jejunum and adrenal gland being the ones with the highest amount of superoxide. The antimalarial drugs studied showed specific behavior according to the type of human tissue analyzed, with atovaquone and quinidine producing the highest percentage of superoxide formation, and doxycycline the lowest. In conclusion, the analysis of superoxide production evaluated in cell membranes of a collection of human tissues allowed for the characterization of the safety profile of these antimalarial drugs against toxicity mediated by oxidative stress.

Antimalarial Atovaquone May Improve Lung Cancer Treatment Outcomes

Antimalarial Atovaquone May Improve Lung Cancer Treatment Outcomes

Inhibition Mechanism of Antimalarial Drugs Targeting the Cytochrome bc1 Complex

Plasmodium falciparum (P. falciparum) is the main parasite known to cause malaria in humans. The antimalarial drug atovaquone is known to inhibit the Qo-site of the cytochrome bc1 complex of P. falciparum, which ultimately blocks ATP synthesis, leading to cell death. Through the years, mutations of the P. falciparum cytochrome bc1 complex, causing resistance to atovaquone, have emerged. The present investigation applies molecular dynamics (MD) simulations to study how the specific mutations Y279S and L282V, known to cause atovaquone resistance in malarial parasites, affect the inhibition mechanism of two known inhibitors. Binding free energy estimates were obtained through free energy perturbation calculations but were unable to confidently resolve the effects of mutations due to the great complexity of the binding environment. Meanwhile, basic mechanistic considerations from the MD simulations provide a detailed characterization of inhibitor binding modes and indicate that the Y279S mutation weakens the natural binding of the inhibitors, while no conclusive effect of the L282V mutation could be observed.

Mitochondrial Inhibitor Atovaquone Increases Tumor Oxygenation and Inhibits Hypoxic Gene Expression in Patients with Non-Small Cell Lung Cancer.

Tumor hypoxia fuels an aggressive tumor phenotype and confers resistance to anticancer treatments. We conducted a clinical trial to determine whether the antimalarial drug atovaquone, a known mitochondrial inhibitor, reduces hypoxia in non-small cell lung cancer (NSCLC). Patients with NSCLC scheduled for surgery were recruited sequentially into two cohorts: cohort 1 received oral atovaquone at the standard clinical dose of 750 mg twice daily, while cohort 2 did not. Primary imaging endpoint was change in tumor hypoxic volume (HV) measured by hypoxia PET-CT. Intercohort comparison of hypoxia gene expression signatures using RNA sequencing from resected tumors was performed. Thirty patients were evaluable for hypoxia PET-CT analysis, 15 per cohort. Median treatment duration was 12 days. Eleven (73.3%) atovaquone-treated patients had meaningful HV reduction, with median change -28% [95% confidence interval (CI), -58.2 to -4.4]. In contrast, median change in untreated patients was +15.5% (95% CI, -6.5 to 35.5). Linear regression estimated the expected mean HV was 55% (95% CI, 24%-74%) lower in cohort 1 compared with cohort 2 (P = 0.004), adjusting for cohort, tumor volume, and baseline HV. A key pharmacodynamics endpoint was reduction in hypoxia-regulated genes, which were significantly downregulated in atovaquone-treated tumors. Data from multiple additional measures of tumor hypoxia and perfusion are presented. No atovaquone-related adverse events were reported. This is the first clinical evidence that targeting tumor mitochondrial metabolism can reduce hypoxia and produce relevant antitumor effects at the mRNA level. Repurposing atovaquone for this purpose may improve treatment outcomes for NSCLC.

The anti-malarial drug atovaquone potentiates platinum-mediated cancer cell death by increasing oxidative stress

Platinum chemotherapies are highly effective cytotoxic agents but often induce resistance when used as monotherapies. Combinatorial strategies limit this risk and provide effective treatment options for many cancers. Here, we repurpose atovaquone (ATQ), a well-tolerated & FDA-approved anti-malarial agent by demonstrating that it potentiates cancer cell death of a subset of platinums. We show that ATQ in combination with carboplatin or cisplatin induces striking and repeatable concentration- and time-dependent cell death sensitization in vitro across a variety of cancer cell lines. ATQ induces mitochondrial reactive oxygen species (mROS), depleting intracellular glutathione (GSH) pools in a concentration-dependent manner. The superoxide dismutase mimetic MnTBAP rescues ATQ-induced mROS production and pre-loading cells with the GSH prodrug N-acetyl cysteine (NAC) abrogates the sensitization. Together, these findings implicate ATQ-induced oxidative stress as key mediator of the sensitizing effect. At physiologically achievable concentrations, ATQ and carboplatin furthermore synergistically delay the growth of three-dimensional avascular spheroids. Clinically, ATQ is a safe and specific inhibitor of the electron transport chain (ETC) and is concurrently being repurposed as a candidate tumor hypoxia modifier. Together, these findings suggest that ATQ is deserving of further study as a candidate platinum sensitizing agent.

Repurposing Atovaquone as a Tumor Hypoxia Modifier: A Window of Opportunity Study in Patients with Resectable Non-small Cell Lung Cancer

Tumor hypoxia confers profound resistance to radiation. An emerging strategy to tackle hypoxia is by reprogramming tumor metabolism. The antimalarial drug atovaquone inhibits oxidative phosphorylation (OXPHOS) and preclinically has been shown to reduce tumor cell oxygen consumption and alleviate tumor hypoxia resulting in radiosensitization. We conducted a translational clinical trial to determine whether atovaquone reduces tumor hypoxia in patients with non-small cell lung cancer (NSCLC). Patients with NSCLC scheduled for surgical resection were recruited sequentially into two cohorts. Cohort 1 received oral atovaquone 750mg bd until the day before surgery. Cohort 2 received no treatment. Tumor hypoxia imaging with 18F-FMISO or 18F-FAZA PET-CT was undertaken at baseline and repeated just prior to resection. Tumor hypoxic volume (HV) was defined using tumor-to-blood tracer uptake ratio ≥1.4, with ≥10% reduction in HV classed a response to treatment. Patients with <1.5 mL HV at baseline were deemed not evaluable for HV analysis and were replaced. Perfusion CT (pCT) and shMOLLI T1 map MRI were undertaken at the same timepoints to assess for changes in tumor perfusion and biochemistry, respectively. An inter-cohort comparison of hypoxia metagene expression signatures obtained by RNA sequencing was conducted from multiple regions per resected tumor. Adverse event monitoring (CTCAE v4.0) was performed until surgery. Forty-six patients were recruited with 30 (15 per cohort) evaluable for paired HV analysis. Patient and tumor characteristics were similar between cohorts. Median duration of atovaquone treatment between imaging timepoints was 12 days. The median change in HV in atovaquone-treated patients was -28% (95% CI: -58.2, -4.4), with 11 (73.3%) patients classed as responders. In contrast, the median change in untreated patients was +15.5% (95% CI: -6.5, +35.5). Using linear regression with pre-surgery HV as outcome adjusting for cohort and baseline HV and tumor volume (log-transformed), the expected geometric mean HV was 60% (95% CI: 30%, 70%) lower for treated patients (p = 0.004). Transcriptomic analysis revealed significantly decreased expression of multiple hypoxia metagene signatures in treated tumors. Altered expression of OXPHOS related pathways was also observed suggesting wider metabolic reprogramming. pCT and MRI results did not show altered tumor perfusion or suggest change in cellularity, respectively, in keeping with an OXPHOS-dependent mechanism of action. No adverse events related to atovaquone were reported. This the first clinical study to investigate whether an OXPHOS inhibitor reduces tumor hypoxia. The data presented supports our pre-clinical finding that atovaquone rapidly reduces tumor hypoxia through decreased oxygen consumption in NSCLC. We will shortly commence phase I clinical testing combining atovaquone with radical chemoradiotherapy in NSCLC.

Potent inhibition of tumour cell proliferation and immunoregulatory function by mitochondria-targeted atovaquone

The FDA-approved prophylactic antimalarial drug atovaquone (ATO) recently was repurposed as an antitumor drug. Studies show that ATO exerts a profound antiproliferative effect in several cancer cells, including breast, ovarian, and glioma. Analogous to the mechanism of action proposed in parasites, ATO inhibits mitochondrial complex III and cell respiration. To enhance the chemotherapeutic efficacy and oxidative phosphorylation inhibition, we developed a mitochondria-targeted triphenylphosphonium-conjugated ATO with varying alkyl side chains (Mito4-ATO, Mito10-ATO, Mito12-ATO, and Mito16-ATO). Results show, for the first time, that triphenylphosphonium-conjugated ATO potently enhanced the antiproliferative effect of ATO in cancer cells and, depending upon the alkyl chain length, the molecular target of inhibition changes from mitochondrial complex III to complex I. Mito4-ATO and Mito10-ATO inhibit both pyruvate/malate-dependent complex I and duroquinol-dependent complex III-induced oxygen consumption whereas Mito12-ATO and Mito16-ATO inhibit only complex I-induced oxygen consumption. Mitochondrial target shifting may have immunoregulatory implications.

Extracellular vesicles derived from Plasmodium-infected and non-infected red blood cells as targeted drug delivery vehicles.

Among several factors behind drug resistance evolution in malaria is the challenge of administering overall doses that are not toxic for the patient but that, locally, are sufficiently high to rapidly kill the parasites. Thus, a crucial antimalarial strategy is the development of drug delivery systems capable of targeting antimalarial compounds to Plasmodium with high specificity. In the present study, extracellular vesicles (EVs) have been evaluated as a drug delivery system for the treatment of malaria. EVs derived from naive red blood cells (RBCs) and from Plasmodium falciparum-infected RBCs (pRBCs) were isolated by ultrafiltration followed by size exclusion chromatography. Lipidomic characterization showed that there were no significant qualitative differences between the lipidomic profiles of pRBC-derived EVs (pRBC-EVs) and RBC-derived EVs (RBC-EVs). Both EVs were taken up by RBCs and pRBCs, although pRBC-EVs were more efficiently internalized than RBC-EVs, which suggested their potential use as drug delivery vehicles for these cells. When loaded into pRBC-EVs, the antimalarial drugs atovaquone and tafenoquine inhibited in vitro P. falciparum growth more efficiently than their free drug counterparts, indicating that pRBC-EVs can potentially increase the efficacy of several small hydrophobic drugs used for the treatment of malaria.

Novel Endochin-Like Quinolones Exhibit Potent In Vitro Activity against Plasmodium knowlesi but Do Not Synergize with Proguanil.

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc1 complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown. We screened several of these novel endochin-like quinolones (ELQs) for their activity against P. knowlesiin vitro and compared this with their activity against P. falciparum tested under identical conditions. We demonstrated that ELQs are potent against P. knowlesi (50% effective concentration, <117 nM) and equally effective against P. falciparum We then screened selected quinolones and partner drugs using a longer exposure (2.5 life cycles) and found that proguanil is 10-fold less potent against P. knowlesi than P. falciparum, while the quinolones demonstrate similar potency. Finally, we used isobologram analysis to compare combinations of the ELQs with either proguanil or atovaquone. We show that all quinolone combinations with proguanil are synergistic against P. falciparum However, against P. knowlesi, no evidence of synergy between proguanil and the quinolones was found. Importantly, the combination of the novel quinolone ELQ-300 with atovaquone was synergistic against both species. Our data identify potentially important species differences in proguanil susceptibility and in the interaction of proguanil with quinolones and support the ongoing development of novel quinolones as potent antimalarials that target multiple species.

Effect of drug dose and timing of treatment on the emergence of drug resistance in vivo in a malaria model.

Background and objectivesThere is a significant interest in identifying clinically effective drug treatment regimens that minimize the de novo evolution of antimicrobial resistance in pathogen populations. However, in vivo studies that vary treatment regimens and directly measure drug resistance evolution are rare. Here, we experimentally investigate the role of drug dose and treatment timing on resistance evolution in an animal model.MethodologyIn a series of experiments, we measured the emergence of atovaquone-resistant mutants of Plasmodium chabaudi in laboratory mice, as a function of dose or timing of treatment (day post-infection) with the antimalarial drug atovaquone.ResultsThe likelihood of high-level resistance emergence increased with atovaquone dose. When varying the timing of treatment, treating either very early or late in infection reduced the risk of resistance. When we varied starting inoculum, resistance was more likely at intermediate inoculum sizes, which correlated with the largest population sizes at time of treatment.Conclusions and implications(i) Higher doses do not always minimize resistance emergence and can promote the emergence of high-level resistance. (ii) Altering treatment timing affects the risk of resistance emergence, likely due to the size of the population at the time of treatment, although we did not test the effect of immunity whose influence may have been important in the case of late treatment. (iii) Finding the ‘right’ dose and ‘right’ time to maximize clinical gains and limit resistance emergence can vary depending on biological context and was non-trivial even in our simplified experiments.Lay summaryIn a mouse model of malaria, higher drug doses led to increases in drug resistance. The timing of drug treatment also impacted resistance emergence, likely due to the size of the population at the time of treatment.