Acridones Are Highly Potent Inhibitors of Toxoplasma gondii Tachyzoites.
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ABSTRACT:
SUBMITTER: Alday PH
PROVIDER: S-EPMC8273083 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Toxoplasma gondii tachyzoites were loaded with the fluorescent indicator fura 2 to investigate the transport mechanisms involved in maintaining their intracellular Ca2+ homoeostasis. The mitochondrial ATPase inhibitor oligomycin and the endoplasmic-reticulum Ca(2+)-ATPase inhibitor thapsigargin increased the intracellular Ca2+ concentration ([Ca2+]i), thus indicating the requirement for ATP and the involvement of the endoplasmic reticulum in maintaining intracellular Ca2+ homoeostasis. The effect of thapsigargin was more accentuated in the presence of extracellular Ca2+, clearly showing that, as occurs with other eukaryotic cells, depletion of intracellular Ca2+ pools led to an increase in the uptake of Ca2+ from the extracellular medium. In addition to these results, we found evidence that, in contrast with what occurs in mammalian cells, T. gondii tachyzoites possess a significant amount of Ca2+ stored in an acidic compartment, termed the acidocalcisome, as indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1 (a specific inhibitor of H(+)-ATPases), nigericin (a K+/H+ exchanger) or the weak base NH4Cl, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry; and (2) the effect of ionomycin, a Ca(2+)-releasing ionophore that cannot take Ca2+ out of acidic organelles and that was more effective after alkalinization of these compartments by addition of bafilomycin A1, nigericin or NH4Cl. Considering the relative importance of the ionomycin-releasable and the ionomycin + NH4Cl-releasable Ca2+ pools, it is apparent that T. gondii tachyzoites contain a significant amount of Ca2+ stored in acidocalcisomes.
| S-EPMC1216957 | biostudies-other
Project description:We have prepared 23 new dehydroartemisinin (DART) trioxane derivatives (11 thiazoles, 2 oxadiazoles, and 10 carboxamides) and have screened them for in vitro activity in the Toxoplasma lytic cycle. Fifteen (65%) of the derivatives were noncytotoxic to host cells (TD(50) > or = 320 microM). Eight thiazole derivatives and two carboxamide derivatives displayed effective inhibition of Toxoplasma growth (IC(50) = 0.25-0.42 microM), comparable in potency to artemether (IC(50) = 0.31 microM) and >100 times more inhibitory than the currently employed front-line drug trimethoprim (IC(50) = 46 microM). The thiazoles as a group were more effective than the other derivatives at inhibiting growth of extracellular as well as intracellular parasites. Unexpectedly, two thiazole trioxanes (5 and 6) were parasiticidal; both inhibited parasite replication irreversibly after parasite exposure to 10 microM of drug for 24 h, whereas the standard trioxane drugs artemisinin and artemether were not parasiticidal. Some of the new derivatives of artemisinin described here represent effective anti-Toxoplasma trioxanes as well as molecular probes for elucidating the mechanism of action of the DART class of artemisinin derivatives.
| S-EPMC2865576 | biostudies-literature
Project description:The triazine WR99210 [4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine] inhibits Toxoplasma gondii in vitro at nanomolar levels (P < 0.05). The 50% inhibitory concentration (IC(50)) was approximately 50 nM. It is a potent inhibitor in vitro and is also effective in vivo. Administration of WR99210 parenterally (i.e., intraperitoneally) reduced the mean number of RH strain tachyzoites present in peritoneal fluid substantially 4 days after intraperitoneal infection of mice. There was a mean of approximately 35 million parasites in control mice as contrasted with approximately 2 million parasites in mice treated with 1.25 mg WR99210/kg of body weight in a representative experiment (P < 0.05). In addition the prodrug PS-15 N'-[3-(2,4, 5-trichlorophenoxy)propyloxy]-N9-(1-methylethyl) imidocarbonimidicdiamide is converted to 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine in vivo when the prodrug is administered orally. PS-15 administered by gavage also reduced intraperitoneal RH strain T. gondii tachyzoite numbers. WR99210 has high efficacy and relatively low toxicity because of its substantial effect on T. gondii dihydrofolate reductase (DHFR) but not the mammalian host DHFR. Amino acid sequences of T. gondii, Plasmodium falciparum, and Homo sapiens DHFRs were compared. It is of interest that of the DHFR amino acids considered to be interacting with WR99210 in P. falciparum within interatomic distances within 3 to 5 A, four of eight were shared with T. gondii DHFR. H. sapiens also shared four amino acids thought to be interacting with WR99210. Efficacy of intraperitoneal administration of WR99210 and peroral administration of PS-15 demonstrate the potential usefulness of this class of compounds in treatment of toxoplasmosis administered either parenterally or perorally. The recent development program for this class of antimicrobials as antimalarials makes our proof of principle of improved efficacy of triazines (compared with the gold standard treatment, pyrimethamine) against T. gondii especially promising.
| S-EPMC1196210 | biostudies-literature
Project description:Drug treatment for toxoplasmosis is problematic, because current drugs cannot eradicate latent infection with Toxoplasma gondii and can cause bone marrow toxicity. Because latent infection remains after treatment, relapse of infection is a problem in both infections in immunocompromised patients and in congenitally infected patients. To identify lead compounds for novel drugs against Toxoplasma gondii, we screened a chemical compound library for anti-Toxoplasma activity, host cell cytotoxicity, and effect on bradyzoites. Of 878 compounds screened, 83 demonstrated >90% parasite growth inhibition. After excluding compounds that affected host cell viability, we further characterized two compounds, tanshinone IIA and hydroxyzine, which had IC50 values for parasite growth of 2.5 ?M and 1.0 ?M, respectively, and had no effect on host cell viability at 25 ?M. Both tanshinone IIA and hydroxyzine inhibited parasite replication after invasion and both reduced the number of in vitro-induced bradyzoites, whereas, pyrimethamine, the current therapy, had no effect on bradyzoites. Both tanshinone IIA and hydroxyzine are potent lead compounds for further medicinal chemistry. The method presented for evaluating compounds for bradyzoite efficacy represents a new approach to the development of anti-Toxoplasma drugs to eliminate latency and treat acute infection.
| S-EPMC5469451 | biostudies-literature
Project description:The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread protozoan parasite of great medical interest. The effect of KP treatment and its potential mechanism of action on T. gondii were evaluated by various methods, including light microscopy, quantitative PCR, flow cytometry, confocal microscopy, and transmission electron microscopy. In the presence of KP, the number of T. gondii tachyzoites able to invade Vero cells and the parasite intracellular proliferation were significantly reduced. Morphological observation and analysis of apoptotic markers suggested that KP is able to trigger an apoptosis-like cell death in T. gondii. Overall, our results indicate that KP could be a promising candidate for the development of new anti-Toxoplasma drugs with a novel mechanism of action.
| S-EPMC5920037 | biostudies-literature
Project description:AIM: To examine three lineages of Toxoplasma gondii RH strain in terms of performance in the dye test, culture, and gene expression. METHODS: Historical data (culture growth and performance in the dye test) from three lineages of RH strain tachyzoites (B, J, and Q) that had been continuously cultured in HeLa cells was assessed. Tachyzoite harvests obtained during continuous cell culture were retrieved from liquid nitrogen and cultured in HeLa cells, providing mRNA that was extracted and used to study gene expression using random amplified polymorphic DNA analysis at different stages of lineage adaptation to continuous culture. RESULTS: The B and Q lineages consistently produced tachyzoites that were successfully used in the dye test and their gene expression was stable after multiple passages. The J lineage had unpredictable growth, tachyzoites unsuitable for use in the dye test, and changing gene expression with multiple passage. CONCLUSION: This study has explained some anomalies in the performance of different stocks of T gondii, and suggests that lineages that are still evolving in cell culture should be avoided.
| S-EPMC1770330 | biostudies-other
Project description:Here, we show that spiroindolone, an effective treatment for plasmodia, is also active against Toxoplasma gondii tachyzoites. In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1?M) and not toxic to human cells at ?10?M. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P=0.002), measured 3 days after the last dose. This inhibition of T. gondii tachyzoites in vitro and in vivo indicates that spiroindolone is a promising lead candidate for further medicine development.
| S-EPMC3957861 | biostudies-literature
Project description:Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.
| S-EPMC10102396 | biostudies-literature
Project description:Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07+/-0.06; n=5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3(-) but was significantly decreased in a dose-dependent manner by low concentrations of N, N'-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.
| S-EPMC1219216 | biostudies-other
Project description:A series of 4-arylthiosemicarbazides substituted at the N1 position with a 5-membered heteroaryl ring was synthesized and evaluated in vitro for T. gondii inhibition proliferation and host cell cytotoxicity. At non-toxic concentrations for the host cells all studied compounds displayed excellent anti-parasitic effects when compared to sulfadiazine, indicating a high selectivity of their anti-T. gondii activity. The differences in bioactivity investigated by DFT calculations suggest that the inhibitory activity of 4-aryl-thiosemicarbazides towards T. gondii proliferation is connected with the electronic structure of the molecule. Further, these compounds were tested as potential antibacterial agents. No growth-inhibiting effect on any of the test microorganisms was observed for all the compounds, even at high concentrations.
| S-EPMC6290556 | biostudies-literature