Potent hydroxamate-derived compounds arrest endodyogeny of Toxoplasma gondii tachyzoites.

Toxoplasmosis is a zoonosis that is a worldwide health problem, commonly affecting fetal development and immunodeficient patients. Treatment is carried out with a combination of pyrimethamine and sulfadiazine, which can cause cytopenia and intolerance and does not lead to a parasitological cure of the infection. Lysine deacetylases (KDACs), which remove an acetyl group from lysine residues in histone and non-histone proteins are found in the Toxoplasma gondii genome. Previous work showed the hydroxamate-type KDAC inhibitors Tubastatin A (TST) and Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) were effective against T. gondii. In the present study, the effects of three hydroxamates (KV-24, KV-30, KV-46), which were originally designed to inhibit human KDAC6, showed different effects against T. gondii. These compounds contain a heterocyclic cap group and a benzyl linker bearing the hydroxamic acid group in para-position. All compounds showed selective activity against T. gondii proliferation, inhibiting tachyzoite proliferation with IC50 values in a nanomolar range after 48h treatment. Microscopy analyses showed that after treatment, tachyzoites presented mislocalization of the apicoplast, disorganization of the inner membrane complex, and arrest in the completion of new daughter cells. The number of dividing cells with incomplete endodyogeny increased significantly after treatment, indicating the compounds can interfere in the late steps of cell division. The results obtained in this work that these new hydroxamates should be considered for future in vivo tests and the development of new compounds for treating toxoplasmosis.

An alternative method to establish an early acute ocular toxoplasmosis model for experimental tests.

PURPOSE: To provide a simple alternative acute ocular toxoplasmosis model with great reproducibility for experimental tests that demand monitoring of the ocular lesion. METHODS: ME49-wt and ME49-GFP tachyzoites from cell culture were used to infect male C57BL6 mice by intraperitoneal injection. B1 expression by real-time polymerase chain reaction (qPCR) assay was used to detect the presence of T. gondii in ocular tissue at the beginning of the infection. Fluorescence microscopy and histopathology analysis were carried out to assess the evolution of the acute infection up to 20 days in both eyes of infected mice. RESULTS: All mice infected with the 104 tachyzoites showed B1 expression in the retina of both eyes, in the RPE (retinal pigment epithelium), and choroid structures, after 5 days of infection. Tachyzoites of the ME49-GFP strain were easily detected by fluorescence microscopy in the retina tissue of mice after 5 days post-infection. After 20 days, mice inflammatory cell infiltrates and a disorganized morphology of the retinal laminar architecture were observed. CONCLUSION: Infection of C57BL6 mice via intraperitoneal with 104 tachyzoites of the ME49-GFP strain from cell culture is a suitable model for acute ocular toxoplasmosis. This model has great reproducibility in establishing the ocular lesion since day 5 post-infection. This model can be suitable for experimental tests of chemotherapy and the investigation of the role of the immune response on the development of uveitis.

Dichloroacetate and Pyruvate Metabolism: Pyruvate Dehydrogenase Kinases as Targets Worth Investigating for Effective Therapy of Toxoplasmosis.

Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite's metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii's in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA.IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.

HDAC inhibitors Tubastatin A and SAHA affect parasite cell division and are potential anti-Toxoplasma gondii chemotherapeutics.

The redirectioning of drugs in the pharmaceutical market is a well-known practice to identify new therapies for parasitic diseases. The histone deacetylase inhibitors Tubastatin A (TST) and Suberoylanilide Hydroxamic Acid (SAHA), firstly developed for cancer treatment, are effective against protozoa parasites. In this work, we aimed to demonstrate the activity of these drugs as potential agents against Toxoplasma gondii, the causative agent of toxoplasmosis. TST and SAHA were active against different genotypes of Toxoplasma gondii, such as, RH (type I), EGS (I/III) and ME49 (type II) strains. The IC50 values for the RH strain were 19 ± 1 nM and 520 ± 386 nM for TST and 41 ± 3 nM and 67 ± 36 nM for SAHA, for 24 and 48 h, respectively. Both compounds were highly selective for T. gondii and their anti-proliferative effect was irreversible for 8 days. The calculated selectivity indexes (39 for TST and 30 for SAHA) make them lead compounds for the future development of anti-Toxoplasma molecules. Western blotting showed TST led to a significant increase of the nuclear histone H4 and a decrease of H3 acetylation levels. Treatment with 1 µM TST and 0.1 µM SAHA for 48 h decreased the amount of global α-tubulin. Fluorescence and electron microscopy showed that both drugs affected the endodyogeny process impairing the budding of daughter cells. The drugs led to the formation of large, rounded masses of damaged parasites with several centrosomes randomly dispersed and incorrect apicoplast division and positioning. TST-treated parasites showed a rupture of the mitochondrial membrane potential and led to a failure of the IMC assembling of new daughter cells. SAHA and TST possibly inhibit HDAC3 and other cytoplasmic or organelle targeted HDACs involved in the modification of proteins other than histones.