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Pimozide inhibits parasite invasion and replication. (A) Host cell monolayers were pretreated with 5 M pimozide (Pmz) or DMSO for 1 h prior to infection with RH-Gal-GFP parasites for 1 h. The cells were fixed without permeabilization and stained with DAPI (to identify host cell nuclei) and anti-SAG1 antiserum. Invasion events were scored via differential staining as GFP SAG1 (invaded) and GFP SAG1 (extracellular). A minimum of 500 host cells were counted for each duplicate sample. Shown are the average values and standard deviations from one experiment representative of three independent experiments performed in duplicate. (B) DMSO-or pimozide-treated extracellular RH-Gal-GFP tachyzoites were incubated in the absence or presence of 1% ethanol (EtOH) for 2 min at 37°C. The supernatants (sMIC2) and parasites (cMIC2) were Western blotted to detect MIC2.-Gal was detected as a loading control. Quantification of sMIC from three independent experiments was performed. (C) The percentage of intracellular parasites from panel B was calculated to determine invasion efficiency. (D) The total numbers of parasites per host cell in DMSO-and pimozide-treated cells were compared. (E) RH parasites were allowed to invade HFFs, and 2 h later, DMSO or pimozide was added. After 24 and 48 h, the cells were fixed and stained with anti-SAG1 antiserum and the number of parasites per vacuole was determined. *, P < 0.05 (unpaired Student t test). MN, multinucleated; AN, anucleated.
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The urgent need to develop new antimicrobial therapies has spawned the development of repurposing screens in which well-studied drugs and other types of compounds are tested for potential off-label uses. As a proof-of-principle screen to identify compounds effective against Toxoplasma gondii, we screened a collection of 1,120 compounds for the abil...
Citations
... Treatment choices included pyrimethamine ® , sulfadiazine ® , atovaquone ® , and clindamycin ® (Romand et al, 1993). But, these drugs were more or less with many side effects and didn't eradicate the bradyzoites (Dittmar et al, 2016). ...
... Finally, it must be emphasized that several drugs used in glaucoma treatment [633][634][635] have antitoxoplasmic activity, including statins [296,642,[684][685][686][687], antihistaminic agents hydroxyzine, clemastine [650,688], ketotifen [689], adrenergics [676,677], sulfonamides (sulfadiazine, sulfamethoxazole, sulfadoxine) [678][679][680], antipsychiatric agents [646,690] (nb. this group of drugs showed direct correlation between clinical potency and presynaptic influence on dopamine neurons [691]), immunosuppressants, immunomodulators, anticancer drugs [680]. ...
Flammer syndrome (FS) is a cluster of signs and symptoms that occurs in a general population regarded usually as healthy individuals. There is a relationship between FS and Raynaud's phenomenon (RP). Chronic latent T. gondii infection is probably responsible for development of RP as well as FS because they share many concomitant diseases, and several drugs used in the treatment of both these clinical entities have antitoxoplasmic activity. Since this ubiquitous parasite is widely distributed in the world more attention should be paid especially in cases with these entities and uncertain diagnosis and not effective treatment.
... Fortunately, transgenic T. gondii strains expressing E. coli beta-galactosidase [39,40] or yellow fluorescent protein [41] are better tools. Consequently, screenings [42] have been performed -either on open-source libraries with the mind of drug repurposing [43,44] or with original compounds, e.g. ruthenium complexes [45][46][47]. ...
Introduction:
Toxoplasmosis constitutes a challenge for public health, animal production and welfare. So far, only a limited panel of drugs has been marketed for clinical applications. Besides classical screening, the investigation of unique targets of the parasite may lead to the identification of novel drugs.
Areas covered:
Herein, the authors describe the methodology to identify novel drug targets in Toxoplasma gondii and review the literature with a focus on the last two decades.
Expert opinion:
During the last two decades, the investigation of essential proteins of T. gondii as potential drug targets has fostered the hope to identify novel compounds for the treatment of toxoplasmosis. Despite good efficacies in vitro, only a few classes of these compounds are effective in suitable rodent models, and none has cleared the hurdle to applications in humans. This shows that target-based drug discovery is in no way better than classical screening approaches. In both cases, off-target effects and adverse side effects in hosts must be considered. Proteomics-driven analyses of parasite- and host-derived proteins that physically bind drug candidates may constitute a suitable tool to characterize drug targets, irrespectively of the drug discovery methods.
... The emerging evidence indicates that testing a variety of substances for possible antiparasitic action is a viable strategy for identifying new treatment options for infectious diseases [1,7]. Recently, we screened chemical libraries containing 1143 compounds and discovered 32 "hit" compounds that inhibited T. gondii growth in vitro [6]. ...
Currently, toxoplasmosis affects nearly one-third of the world’s population, but the available treatments have several limitations. This factor underscores the search for better therapy for toxoplasmosis. Therefore, in the current investigation, we investigated the potential of emodin as a new anti-Toxoplasma gondii while exploring its anti-parasitic mechanism of action. We explored the mechanisms of action of emodin in the presence and absence of an in vitro model of experimental toxoplasmosis. Emodin showed strong anti-T. gondii action with an EC50 value of 0.03 µg/mL; at this same effective anti-parasite concentration, emodin showed no appreciable host cytotoxicity. Likewise, emodin showed a promising anti-T. gondii specificity with a selectivity index (SI) of 276. Pyrimethamine, a standard drug for toxoplasmosis, had an SI of 2.3. The results collectively imply that parasite damage was selective rather than as a result of a broad cytotoxic effect. Furthermore, our data confirm that emodin-induced parasite growth suppression stems from parasite targets and not host targets, and indicate that the anti-parasite action of emodin precludes oxidative stress and ROS production. Emodin likely mediates parasite growth suppression through means other than oxidative stress, ROS production, or mitochondrial toxicity. Collectively, our findings support the potential of emodin as a promising and novel anti-parasitic agent that warrants further investigation.
... This protocol was thus adopted in automated image analysis, which resulted in the identification of seven kinase inhibitors that mostly inhibited p38-MAP kinase, and two of which-Cpd2 (SB 203580) and Cpd3 (SB 208)-were previously reported to reduce T. gondii growth (Wei et al., 2002;Wiley et al., 2010). It should be emphasized that the compound library used in our analyses was used by Dittmar et al. (2016) to assess inhibition of T. gondii growth based on b-galactosidase assay and these authors identified 94 compounds and a significant number of small molecules known to impact dopamine or estrogen signaling. Among the 12 compounds identified in our screen, four inhibitors (Cpd4, Cpd6, Cpd9 and Cpd12) were not present in the hits reported by Dittmar et al. (2016). ...
... It should be emphasized that the compound library used in our analyses was used by Dittmar et al. (2016) to assess inhibition of T. gondii growth based on b-galactosidase assay and these authors identified 94 compounds and a significant number of small molecules known to impact dopamine or estrogen signaling. Among the 12 compounds identified in our screen, four inhibitors (Cpd4, Cpd6, Cpd9 and Cpd12) were not present in the hits reported by Dittmar et al. (2016). In addition, one of the compounds identified in our study (Cpd12, ZK 164015) as an estrogen receptor antagonist has not been identified in the aforementioned screening. ...
Apicomplexa phylum includes numerous obligate intracellular protozoan parasites that are life threatening for humans and animals. In this context, Plasmodium falciparum and Toxoplasma gondii are of particular interest, as they are responsible for malaria and toxoplasmosis, respectively, for which efficient vaccines are presently lacking and therapies need to be improved. Apicomplexan parasites have a highly polarized morphology, with their apical end containing specific secretory organelles named rhoptries and micronemes, which depend on the unique receptor and transporter sortilin TgSORT for their biogenesis. In the present study, we took advantage of the subcellular polarity of the parasite to engineer a clonal transgenic Toxoplasma line that expresses simultaneously the green fluorescent protein TgSORT-GFP in the post-Golgi-endosome-like compartment and the red fluorescent protein rhoptry ROP1-mCherry near the apical end. We utilized this fluorescent transgenic T. gondii to develop a miniaturized image-based phenotype assay coupled to an automated image analysis. By applying this methodology to 1,120 compounds, we identified 12 that are capable of disrupting the T. gondii morphology and inhibiting intracellular replication. Analysis of the selected compounds confirmed that all 12 are kinase inhibitors and intramembrane pumps, with some exhibiting potent activity against Plasmodium falciparum. Our findings highlight the advantage of comparative and targeted phenotypic analysis involving two related parasite species as a means of identifying molecules with a conserved mode of action.
... In addition, it was reported that tamoxifen had a direct antibacterial effect on Mtb, synergizing with first-line TB antibiotics (29,30). In contrast to these reported direct antimicrobial effects, there is evidence that the inhibitory effect of tamoxifen on intracellular Toxoplasma growth is mediated in a hostdirected manner by inducing autophagic degradation of the parasite-containing vacuole (31). However, the role of tamoxifen-induced autophagy and possibly other tamoxifenmodulated host pathways in controlling Mtb or other bacterial infections remains incompletely defined. ...
... Tamoxifen treatment of noninfected larvae caused differential expression of 141 genes, including genes involved in estrogen receptor (ER) signaling and autophagy and other cellular stress pathways, consistent with known effects of tamoxifen exposure (Supplemental Data S1 at https://doi.org/10.5281/zenodo.5788543) (24,25,31,(48)(49)(50). ...
... Tamoxifen treatment modulates autophagy in infected human macrophages and zebrafish. Because tamoxifen induces and modulates autophagy and because autophagy contributes to host defense against TB, we next investigated the role of tamoxifen-induced autophagy in inhibiting bacterial outgrowth (31,50,64). We first used Cyto-ID, a tracer for autophagy-related vesicles offering the advantage of staining all intracellular FIG 3 Legend (Continued) [vol/vol]) starting at 1 hpi. ...
Tuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis . This pathogen evades the immune defenses of its host and grows intracellularly in immune cells, particularly inside macrophages.
... Cell line preparation [11] : The Vero cells were provided from VACSERA company, Egypt, and maintained in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ ml Penicillin, and 100 μg/ml Streptomycin (Sigma-Aldrich, USA) in 75 cm 2 flasks, and incubated in 5% CO 2 at 37°C [10] . The in vitro study was performed at the International Center for Training and Advanced Research (ICTAR, Egypt), Al-Azhar University, Cairo, Egypt. ...
... Their results were attributed to a process called autophagy, in which there is damage of mitochondria and cell membrane. Dittmar et al. [10] also explained that Tamoxifen could reduce the parasite burden through the same process. ...
... Despite the importance of toxoplasmosis to public health, considering its high prevalence in the human population and the serious clinical manifestations, mainly in immunocompromised patients and in cases of congenital infection [5], there are still very few therapeutic options available, these being effective only against the acute form of the disease [6]. ...
Toxoplasma gondii is a protozoan that infects up to a third of the world’s population. This parasite can cause serious problems, especially if a woman is infected during pregnancy, when toxoplasmosis can cause miscarriage, or serious complications to the baby, or in an immunocompromised person, when the infection can possibly affect the patient’s eyes or brain. To identify potential drug candidates that could counter toxoplasmosis, we selected 13 compounds which were pre-screened in silico based on the proteome of T. gondii to be evaluated in vitro against the parasite in a cell-based assay. Among the selected compounds, three demonstrated in vitro anti-T. gondii activity in the nanomolar range (almitrine, bortezomib, and fludarabine), and ten compounds demonstrated anti-T. gondii activity in the micromolar range (digitoxin, digoxin, doxorubicin, fusidic acid, levofloxacin, lomefloxacin, mycophenolic acid, ribavirin, trimethoprim, and valproic acid). Almitrine demonstrated a Selectivity Index (provided by the ratio between the Half Cytotoxic Concentration against human foreskin fibroblasts and the Half Effective Concentration against T. gondii tachyzoites) that was higher than 47, whilst being considered a lead compound against T. gondii. Almitrine showed interactions with the Na+/K+ ATPase transporter for Homo sapiens and Mus musculus, indicating a possible mechanism of action of this compound.
... The recruitment of LC3 and ATG8 orthologs on PVM is necessary for autophagosome formation, and ATG8 labelled with a green fluorescent protein (GFP) at its amino terminus has emerged as a useful indicator of autophagy levels (Besteiro et al., 2011;Subauste, 2021). Increasing the combination of LC3-GFP and PV limits the growth of parasites by inducing the autophagic degradation of PV (Dittmar et al., 2016). Varberg et al. found that a high concentration of Plasmodium ATG3-ATG8 interaction inhibitors can promote the lipidation of ATG8, thereby stimulating autophagy in T. gondii-infected cells. ...
Toxoplasma gondii infection is a severe health threat that endangers billions of people worldwide. T. gondii utilizes the host cell membrane to form a parasitophorous vacuole (PV), thereby fully isolating itself from the host cell cytoplasm and making intracellular clearance difficult. PV can be targeted and destroyed by autophagy. Autophagic targeting results in T. gondii killing via the fusion of autophagosomes and lysosomes. However, T. gondii has developed many strategies to suppress autophagic targeting. Accordingly, the interplay between host cell autophagy and T. gondii is an emerging area with important practical implications. By promoting the canonical autophagy pathway or attenuating the suppression of autophagic targeting, autophagy can be effectively utilized in the development of novel therapeutic strategies against T gondii. Here, we have illustrated the complex interplay between host cell mediated autophagy and T. gondii. Different strategies to promote autophagy in order to target the parasite have been elucidated. Besides, we have analyzed some potential new drug molecules from the DrugBank database using bioinformatics tools, which can modulate autophagy. Various challenges and opportunities focusing autophagy mediated T. gondii clearance have been discussed, which will provide new insights for the development of novel drugs against the parasite.
... Screening of the Prestwick library identified Amiodaron the antiarrhythmic drug to have a fungicidal effect on Cryptococcus neoformans, a drug that is also capable of crossing blood-brain barrier [16]. Due to the growing necessity for the development of new antifungal drugs, particularly those with some with anti-biofilm activity, we performed a high throughput screening of a Tocris 2.0 Micro library that encompasses different categories of compounds to identify their antifungal activity against C. albicans and C. auris, a library that has only been used to screen anti-parasite activity against Toxoplasma gondii [17]. Here, we show for the first time that screening this library has identified a number of key compounds that are capable of inhibiting Candida biofilm formation, either alone or in synergy with conventional antifungal agents. ...
Background and purpose:
Biofilms formed by Candida species present a significant clinical problem due to the ineffectiveness of many conventional antifungal agents, in particular the azole class. We urgently require new and clinically approved antifungal agents quickly for treatment of critically ill patients.
Methods:
To improve efficiency in antifungal drug development, we utilized a library of 1280 biologically active molecules within the Tocriscreen 2.0 Micro library. C. auris NCPF 8973 and C. albicans SC5314 were initially screened for biofilm inhibitory activity using metabolic and biomass quantitative assessment methods, followed up by targeted evaluation of five selected hits.
Results:
The initial screening (80% metabolic inhibition rate) revealed that there was 90 and 87 hits (approx. 7%) for C. albicans and C. auris, respectively. Additionally, all five compounds selected from the initial hits exhibited a biofilm inhibition effect against several key Candida species tested, including C. glabrata and C. krusei. Toyocamycin displayed the most potent activity at concentrations as low as 0.5 µg/mL, though was limited to inhibition. Darapladib demonstrated an efficacy for biofilm inhibition and treatment at a concentration range from 8 to 32 µg/mL and from 16 to 256 µg/mL, respectively. Combinational testing with conventional antifungals against C. albicans strains demonstrated a range of synergies for planktonic cells, and notably an anti-biofilm synergy for darapladib and caspofungin.
Interpretation:
Together, these data provide new insights into antifungal management possibilities for Candida biofilms.
