Atovaquone enhances antitumor efficacy of TCR-T therapy by augmentation of ROS-induced ferroptosis in hepatocellular carcinoma.

T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for tumor treatment, yet hindered by tumor immune evasion resulting in poor therapeutic efficacy. The introduction of ferroptosis-targeted inducers offers a potential solution, as they empower T cells to induce ferroptosis and exert influence over the tumor microenvironment. Atovaquone (ATO) stands as a prospective pharmaceutical candidate with the potential to target ferroptosis, effectively provoking an excessive generation and accumulation of reactive oxygen species (ROS). In this study, we evaluated the effectiveness of a combination therapy comprising ATO and TCR-T cells against hepatocellular carcinoma (HCC), both in vitro and in vivo. The results of lactate dehydrogenase and cytokine assays demonstrated that ATO enhanced cytotoxicity mediated by AFP-specific TCR-T cells and promoted the release of IFN-γ in vitro. Additionally, in an established HCC xenograft mouse model, the combined therapy with low-dose ATO and TCR-T cells exhibited heightened efficacy in suppressing tumor growth, with no apparent adverse effects, comparable to the results achieved through monotherapy. The RNA-seq data unveiled a significant activation of the ferroptosis-related pathway in the combination therapy group in comparison to the TCR-T cells group. Mechanistically, the synergy between ATO and TCR-T cells augmented the release of IFN-γ by TCR-T cells, while concurrently elevating the intracellular and mitochondrial levels of ROS, expanding the labile iron pool, and impairing the integrity of the mitochondrial membrane in HepG2 cells. This multifaceted interaction culminated in the potentiation of ferroptosis within the tumor, primarily induced by an excess of ROS. In summary, the co-administration of ATO and TCR-T cells in HCC exhibited heightened vulnerability to ferroptosis. This heightened susceptibility led to the inhibition of tumor growth and the stimulation of an anti-tumor immune response. These findings suggest that repurposing atovaquone for adoptive cell therapy combination therapy holds the potential to enhance treatment outcomes in HCC.

Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine.

BACKGROUND: To gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model. METHODS: The animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104 P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge. RESULTS: Group 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow. CONCLUSIONS: This research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing P. vivax.

A Smartphone App for Real-Time Assessment of Malaria Prophylaxis Adverse Events.

Background: Growth of international travel to malarial areas over the last decades has contributed to more travelers taking malaria prophylaxis. Travel-related symptoms may be wrongly attributed to malaria prophylaxis and hinder compliance. Here, we aimed to assess the frequency of real-time reporting of symptoms by travelers following malaria prophylaxis using a smartphone app. Method: Adult international travelers included in this single-center study (Barcelona, Spain) used the smartphone Trip Doctor® app developed by our group for real-time tracking of symptoms and adherence to prophylaxis. Results: Six hundred four (n = 604) international travelers were included in the study; 74.3% (449) used the app daily, and for one-quarter of travelers, malaria prophylaxis was prescribed. Participants from the prophylaxis group traveled more to Africa (86.7% vs. 4.3%; p < 0.01) and to high travel medical risk countries (60.8% vs. 18%; p < 0.01) and reported more immunosuppression (30.8% vs. 23.1% p < 0.01). Regarding symptoms, no significant intergroup differences were observed, and no relationship was found between the total number of malarial pills taken and reported symptoms. Conclusions: In our cohort, the number of symptoms due to malaria prophylaxis was not significantly higher than in participants for whom prophylaxis was not prescribed, and the overall proportion of symptoms is higher compared with other studies.

Potential Anti-Mpox Virus Activity of Atovaquone, Mefloquine, and Molnupiravir, and Their Potential Use as Treatments.

BACKGROUND: Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. METHODS: We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. RESULTS: Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 µM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. CONCLUSIONS: These data suggest that atovaquone would be a potential candidate for treating mpox.

Atovaquone inhibits colorectal cancer metastasis by regulating PDGFRß/NF-κB signaling pathway.

BACKGROUND: Colorectal cancer is a common malignant tumour. Invasive growth and distant metastasis are the main characteristics of its malignant biological behaviour, and they are also the primary factors leading to death in colon cancer patients. Atovaquone is an antimalarial drug, and its anticancer effect has recently been demonstrated in several cancer models in vitro and in vivo, but it has not been examined in the treatment of colorectal cancer. METHODS: To elucidate the effect of atovaquone on colorectal cancer. We used RNA transcriptome sequencing, RT‒PCR and Western blot experiments to examine the expression of NF-κB (p-P65), EMT-related proteins and related inflammatory factors (IL1B, IL6, CCL20, CCL2, CXCL8, CXCL6, IL6ST, FAS, IL10 and IL1A). The effect of atovaquone on colorectal cancer metastasis was validated using an animal model of lung metastases. We further used transcriptome sequencing, the GCBI bioinformatics database and the STRING database to predict relevant target proteins. Furthermore, pathological sections were collected from relevant cases for immunohistochemical verification. RESULTS: This study showed that atovaquone could inhibit colorectal cancer metastasis and invasion in vivo and in vitro, inhibit the expression of E-cadherin protein, and promote the protein expression of N-cadherin, vimentin, ZEB1, Snail and Slug. Atovaquone could inhibit EMT by inhibiting NF-κB (p-P65) and related inflammatory factors. Further bioinformatics analysis and verification showed that PDGFRß was one of the targets of atovaquone. CONCLUSION: In summary, atovaquone can inhibit the expression of NF-κB (p-P65) and related inflammatory factors by inhibiting the protein expression of p-PDGFRß, thereby inhibiting colorectal cancer metastasis. Atovaquone may be a promising drug for the treatment of colorectal cancer metastasis.

Atovaquone exposure and Pneumocystis jirovecii cytochrome b mutations: French data and review of the literature.

Pneumocystis jirovecii is a transmissible fungus responsible for severe pneumonia (Pneumocystis pneumonia [PCP]) in immunocompromised patients. Missense mutations due to atovaquone selective pressure have been identified on cytochrome b (CYB) gene of P. jirovecii. It was recently shown that atovaquone prophylaxis can lead to the selection of specific P. jirovecii CYB mutants potentially resistant to atovaquone among organ transplant recipients. In this context, our objectives were to provide data on P. jirovecii CYB mutants and the putative selective pressure exerted by atovaquone on P. jirovecii organisms in France. A total of 123 patients (124 P. jirovecii specimens) from four metropolitan hospitals and two overseas hospitals were retrospectively enrolled. Fourteen patients had prior exposure to atovaquone, whereas 109 patients did not at the time of P. jirovecii detection. A 638 base-pair fragment of the CYB gene of P. jirovecii was amplified and sequenced. A total of 10 single nucleotide polymorphisms (SNPs) were identified. Both missense mutations C431T (Ala144Val) and C823T (Leu275Phe), located at the Qo active site of the enzyme, were significantly associated with prior atovaquone exposure, these mutations being conversely incidental in the absence of prior atovaquone exposure (P < 0.001). Considering that the aforementioned hospitals may be representative of the national territory, these findings suggest that the overall presence of P. jirovecii CYB mutants remains low in France.

The mutations C431T (Ala144Val) and C823T (Leu275Phe) at the cytochrome b (CYB) active site of Pneumocystis jirovecii are associated with patient prior exposure to atovaquone. Conversely, these mutations are incidental in the absence of exposure. Overall, the presence of P. jirovecii CYB mutants remains low in France.

Combination of Clofazimine and Atovaquone as a Potent Therapeutic Regimen for the Radical Cure of Babesia microti Infection in Immunocompromised Hosts.

Human babesiosis caused by Babesia microti can be fatal in immunocompromised patients, and the currently used drugs are often ineffective. A recent study found that clofazimine clears B. microti Munich strain in immunocompromised mice. In the present study, we investigated the efficacies of clofazimine and 2-drug combinations involving clofazimine, atovaquone, and azithromycin against B. microti Peabody mjr strain in immunocompromised mice. Treatment with clofazimine alone, clofazimine plus azithromycin, and atovaquone plus azithromycin was ineffective and failed to eliminate the parasites completely, while a 44-day treatment with clofazimine plus atovaquone was highly effective and resulted in a radical cure.

Proguanil and atovaquone use is associated with lower colorectal cancer risk: a nationwide cohort study.

BACKGROUND: Individuals with a family history of colorectal cancer (CRC) are at a high risk of developing CRC. Preclinical studies suggest that the anti-malaria drug proguanil and atovaquone might play a role in preventing CRC, but population-based evidence is still lacking. METHODS: By accessing a couple of nationwide Swedish registers, we performed a cohort study to explore whether using proguanil and atovaquone might associate with a lower risk of CRC by adopting a new-user study design. Adults who have 1 or more first-degree relatives (parents or siblings) diagnosed with CRC were identified and linked with the Prescribed Drug Register to evaluate their administration history of proguanil and atovaquone. Survival analysis of the time to CRC diagnosis with Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: A total of 16,817 incident proguanil/atovaquone users were identified and matched with 168,170 comparisons, who did not use proguanil/atovaquone, on the ratio of 1:10. We found a significant negative association between proguanil/atovaquone use and risk of CRC (adjusted HR, 0.76; 95% CI, 0.62-0.93). Test for trend showed significant dose- and duration-response correlations (P < 0.001). The association was more pronounced in CRC diagnosed at an advanced stage than at an early stage (adjusted HR, 0.69 vs.0.81). CONCLUSIONS: This national-wide population-based cohort study showed that the use of proguanil and atovaquone was associated with a reduced risk of CRC among individuals with a family history of CRC.

The effect of atovaquone on the mitochondrial membrane potential of Babesiagibsoni.

Atovaquone (ATV) has a growth inhibitory effect against Babesia gibsoni. The target site is considered mitochondria, as in the case of Plasmodium spp.; ATV would collapse the mitochondrial membrane potential. B. gibsoni has also reported that single nucleotide polymorphisms in cytochrome b of mitochondria are involved in ATV susceptibility. However, the details are still unknown. The study aim was to measure the mitochondrial membrane potential of B. gibsoni and evaluate the effect of ATV alone and combined with proguanil (PG) on the mitochondrial membrane potential. As a result of exposure of wild-type B. gibsoni to ATV alone, the number of cells with decreased mitochondrial membrane potential increased. When wild-type B. gibsoni was exposed to the ATV + PG combination, the peak value of mitochondrial membrane potential was larger than that when exposed to ATV alone. It was suggested that ATV alone affects the mitochondrial membrane potential of B. gibsoni, and the effect is enhanced by the combination of ATV and PG. The effect of ATV was weakened for B. gibsoni having reduced sensitivity to ATV (B. gibsoni with M121I), and the effect was not enhanced by the combination of ATV and PG. Although we still need to elucidate the mechanism of ATV and PG for B. gibsoni, these results strongly suggests that the target of ATV for B. gibsoni is also cytochrome b of mitochondria.

Comparative effectiveness of trimethoprim-sulfamethoxazole versus atovaquone for the prophylaxis of pneumocystis pneumonia in patients with connective tissue diseases receiving prolonged high-dose glucocorticoids.

We compared the prophylactic effect of trimethoprim-sulfamethoxazole (TMP-SMX) with atovaquone for pneumocystis pneumonia (PCP) in patients with connective tissue diseases (CTDs) receiving high-dose glucocorticoids. Patients with CTDs aged ≥ 18 years who were treated with a prolonged course (≥ 4 weeks) of glucocorticoids (≥ 20 mg/day prednisone) in a Japanese tertiary center between 2013 and 2017 were included. The patients were categorized into two groups: TMP-SMX and atovaquone group. Adjusted cumulative incidence of PCP was compared between the two groups after propensity score weighting for differences in confounding factors. A total of 480 patients with a prolonged high-dose glucocorticoid treatment were identified. Out of 383 patients with TMP-SMX prophylaxis, 102 (26.8%) patients experienced adverse events leading to discontinuation within 4 weeks of initiation, while no patient in the atovaquone discontinued the therapy. Two hundred eighty-one patients received TMP-SMX, while 107 received atovaquone for PCP prophylaxis. During a total of 397.0 person-years, 7 PCP cases (2 in the TMP-SMX, 5 in the atovaquone) occurred with a mortality rate of 54.5%. After adjusting for differences in baseline characteristics, the adjusted cumulative incidence of PCP was similar between the two group (HR 0.97, 95% CI 0.19-5.09, p = 0.97). Prophylactic effects for PCP in CTDs patients receiving prolonged high-dose glucocorticoids were similar between TMP-SMX and atovaquone. Atovaquone was well-tolerated with no side effects.

A Novel Encochleated Formulation Improves Atovaquone Activity in a Murine Model of Pneumocystis Pneumonia.

Although atovaquone is effective in treating and preventing Pneumocystis pneumonia (PCP), its use is limited by nonlinear absorption and adverse events. The current study was undertaken to examine the activity of encochleated atovaquone (eATQ), a novel lipid-crystal nanoparticle formulation, in a mouse model of PCP. eATQ 100-200 mg was superior to commercially available atovaquone at 14 days in decreasing total Pneumocystis nuclei and asci. eATQ plus anidulafungin reduced nuclei significantly better than commercial atovaquone plus anidulafungin. eATQ is a novel formulation of atovaquone that warrants further evaluation for treatment and prevention of PCP.

Exploring Ubiquinone Biosynthesis Inhibition as a Strategy for Improving Atovaquone Efficacy in Malaria.

Atovaquone (AV) acts on the malaria parasite by competing with ubiquinol (UQH2) for its union to the mitochondrial bc1 complex, preventing the ubiquinone-8 and ubiquinone-9 (UQ-8 and UQ-9) redox recycling, which is a necessary step in pyrimidine biosynthesis. This study focused on UQ biosynthesis in Plasmodium falciparum and adopted proof-of-concept research to better elucidate the mechanism of action of AV and improve its efficacy. Initially, UQ biosynthesis was evaluated using several radioactive precursors and chromatographic techniques. This methodology was suitable for studying the biosynthesis of both UQ homologs and its redox state. Additionally, the composition of UQ was investigated in parasites cultivated at different oxygen saturations or in the presence of AV. AV affected the redox states of both UQ-8 and UQ-9 homologs by increasing the levels of the respective reduced forms. Conversely, low-oxygen environments specifically inhibited UQ-9 biosynthesis and increased the antimalarial efficacy of AV. These findings encouraged us to investigate the biological importance and the potential of UQ biosynthesis as a drug target based on its inhibition by 4-nitrobenzoate (4-NB), a 4-hydroxybenzoate (4-HB) analog. 4-NB effectively inhibits UQ biosynthesis and enhances the effects of AV on parasitic growth and respiration rate. Although 4-NB itself exhibits poor antimalarial activity, its 50% inhibitory concentration (IC50) value increased significantly in the presence of a soluble UQ analog, p-aminobenzoic acid (pABA), or 4-HB. These results indicate the potential of AV combined with 4-NB as a novel therapy for malaria and other diseases caused by AV-sensitive pathogens.

Selection of Cytochrome b Mutants Is Rare among Plasmodium falciparum Patients Failing Treatment with Atovaquone-Proguanil in Cambodia.

Atovaquone-proguanil remains effective against multidrug-resistant Plasmodium falciparum in Southeast Asia, but resistance is mediated by a single point mutation in cytochrome b (cytb) that can arise during treatment. Among 14 atovaquone-proguanil treatment failures in a clinical trial in Cambodia, only one recrudescence harbored the cytb mutation Y268C. Deep sequencing did not detect the mutation at baseline or in the first 3 days of treatment, suggesting that it arose de novo Further sequencing across cytb similarly found no low-frequency cytb mutations that were up-selected from baseline to recrudescence. Copy number amplification in dihydroorotate dehydrogenase (DHODH) and cytb as markers of atovaquone tolerance was also absent. Cytb mutation played a minor role in atovaquone-proguanil treatment failures in an active comparator clinical trial.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice.

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii.

Toxoplasma gondiiis an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline tonew derivatives improved the anti-T. gondiiactivity 3.4 fold. The most active derivative presented ED50in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.

Atovaquone-HSA nano-drugs enhance the efficacy of PD-1 blockade immunotherapy by alleviating hypoxic tumor microenvironment.

BACKGROUND: Hypoxia is inherent character of most solid malignancies, leading to the failure of chemotherapy, radiotherapy and immunotherapy. Atovaquone, an anti-malaria drug, can alleviate tumor hypoxia by inhibiting mitochondrial complex III activity. The present study exploits atovaquone/albumin nanoparticles to improve bioavailability and tumor targeting of atovaquone, enhancing the efficacy of anti-PD-1 therapy by normalizing tumor hypoxia. METHODS: We prepared atovaquone-loaded human serum albumin (HSA) nanoparticles stabilized by intramolecular disulfide bonds, termed HSA-ATO NPs. The average size and zeta potential of HSA-ATO NPs were measured by particle size analyzer. The morphology of HSA-ATO NPs was characterized by transmission electron microscope (TEM). The bioavailability and safety of HSA-ATO NPs were assessed by animal experiments. Flow cytometry and ELISA assays were used to evaluate tumor immune microenvironment. RESULTS: Our data first verified that atovaquone effectively alleviated tumor hypoxia by inhibiting mitochondrial activity both in vitro and in vivo, and successfully encapsulated atovaquone in vesicle with albumin, forming HSA-ATO NPs of approximately 164 nm in diameter. We then demonstrated that the HSA-ATO NPs possessed excellent bioavailability, tumor targeting and a highly favorable biosafety profile. When combined with anti-PD-1 antibody, we observed that HSA-ATO NPs strongly enhanced the response of mice bearing tumor xenografts to immunotherapy. Mechanistically, HSA-ATO NPs promoted intratumoral CD8+ T cell recruitment by alleviating tumor hypoxia microenvironment, thereby enhancing the efficacy of anti-PD-1 immunotherapy. CONCLUSIONS: Our data provide strong evidences showing that HSA-ATO NPs can serve as safe and effective nano-drugs to enhance cancer immunotherapy by alleviating hypoxic tumor microenvironment.

Histopathological and ultrastructural assessment of atovaquone-proguanil hydrochloride combination in chronic murine toxoplasmosis.

Over one billion people worldwide are expected to have Toxoplasma gondii infection with anonymous health problems. Available therapies are ineffective for persistent chronic toxoplasmosis. So, there is an imperative need for effective therapies to eliminate chronic tissue stage. In this study, we aimed to assess the effect of a drug combination of atovaquone and proguanil hydrochloride in the treatment of experimental chronic toxoplasmosis. Fifty Swiss Webster mice were used in the study. Forty mice were infected with Me49 type II cystogenic Toxoplasma gondii strain and allocated into four groups: infected untreated (vehicle-administered), infected and treated with cotrimoxazole (CTX) 370 mg/kg/day, infected and treated with atovaquone (ATV) 100 mg/kg/day, and infected and treated with atovaquone/proguanil (ATV/PROG) 50 mg/kg/day. An additional group of uninfected mice was used as an uninfected control group. Drug treatment was initiated 8 weeks post-infection and continued for two weeks. All mice were sacrificed 12 weeks post-infection. Parasitological and histopathological parameters were assessed. Toxoplasma gondii cysts recovered from brain tissue homogenates of both infected untreated and ATV/PROG-treated groups were examined by scanning electron microscopy. Combined ATV/PROG treatment demonstrated a significant reduction of Toxoplasma gondii cyst count in brain tissue (a reduction rate of 84.87%) compared to untreated group (P < .001). Brain tissues obtained from ATV/PROG treated group showed reduction of inflammatory infiltrate and marked attenuation and deformation of recovered Toxoplasma gondii cysts. We conclude that ATV/PROG drug combination could offer a potential drug therapy for Toxoplasma gondii chronic cystic stage.

Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells.

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-ß and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-ß, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-ß and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

Atovaquone Inhibits Arbovirus Replication through the Depletion of Intracellular Nucleotides.

Arthropod-borne viruses represent a significant public health threat worldwide, yet there are few antiviral therapies or prophylaxes targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus (ZIKV) in the Americas. One potential avenue to identify new and pregnancy-acceptable antiviral compounds is to repurpose well-known and widely used FDA-approved drugs. In this study, we addressed the antiviral role of atovaquone, an FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides, indicating that atovaquone functions through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner, providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women and children.IMPORTANCE The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy-acceptable drugs that have the potential to mitigate arbovirus infections and disease, yet this has not been explored in detail. In this study, we show that the common antiparasitic drug atovaquone inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease.

In silico, in vitro and in vivo evaluation of natural Bignoniaceous naphthoquinones in comparison with atovaquone targeting the selection of potential antimalarial candidates.

The natural naphthoquinones lapachol, α- and ß-lapachone are found in Bignoniaceous Brazilian plant species of the Tabebuia genus (synonym Handroanthus) and are recognized for diverse bioactivities, including as antimalarial. The aim of the present work was to perform in silico, in vitro and in vivo studies to evaluating the antimalarial potential of these three naphthoquinones in comparison with atovaquone, a synthetic antimalarial. The ADMET properties of these compounds were predicted in silico by the preADMET program. The in vitro toxicity assays were experimentally determined in immortalized and tumoral cells from different organs. In vivo acute oral toxicity was also evaluated for lapachol. Several favorable pharmacokinetics data were predicted although, as expected, high cytotoxicity was experimentally determined for ß-lapachone. Lapachol was not cytotoxic or showed low cytotoxicity to all of the cells assayed (HepG2, A549, Neuro 2A, LLC-PK1, MRC-5), it was nontoxic in the acute oral test and disclosed the best parasite selectivity index in the in vitro assays against chloroquine resistant Plasmodium falciparum W2 strain. On the other hand, α- and ß-lapachone were more potent than lapachol in the antiplasmodial assays but with low parasite selectivity due to their cytotoxicity. The diversity of data here reported disclosed lapachol as a promising candidate to antimalarial drug development.