Valutazione in vitro e in vivo di thiosemicarbazoni contro l’infezione da Toxoplasma gondii

Toxoplasma gondii is a globally widespread apicomplexan parasite, with domestic cats (or felines in general) as definitive hosts and many warm-blooded animals, including humans, as intermediate hosts. The parasite has three infectious stages: tachyzoites, bradyzoites and oocysts. After infection (usually through ingestion of tissue cysts containing bradyzoites), cats shed oocysts in the environment which sporulate and become infectious after several days. Intermediate hosts can be infected through the ingestion of food or water contaminated with sporulated oocysts or by ingestion of tissue cysts. Toxoplasma gondii infections are common in humans, livestock, and companion animals, but clinical disease is rare. Over 80% of immunocompetent individuals, especially in Europe and North America, experience no symptoms. In the remaining cases, mild symptoms like fever and lymphadenopathy may occur. However, in immunocompromised individuals, toxoplasmosis often results from the reactivation of a chronic infection and can be life-threatening. The primary organs affected by T. gondii include the brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes and pancreas. Congenital toxoplasmosis is another significant concern, occurring in pregnant women, where the severity of outcomes depends on the stage of pregnancy during maternal infection. Treatment options for toxoplasmosis are limited, with the folate pathway being the primary target of anti-Toxoplasma drugs, such as pyrimethamine and sulfadiazine. Other drugs, such as atovaquone, spiramycin, azithromycin, and clindamycin are also used as second-line drugs. Unfortunately, current treatment regimens have side effects due to myelotoxicity (not to mention more severe effects that can be life-threatening) and require discontinuation of therapy. In addition, a key challenge in treating T. gondii infections is that, while existing drugs can control acute toxoplasmosis, no approved therapy is available to eliminate the tissue cysts that cause chronic infections and the risk reactivation. For this reason, new compounds have been recently optimized and tested to develop new therapies that address all these issues. Thiosemicarbazones (TSCs) are organosulfur compounds which have been shown to exhibit very good activity against T. gondii. The present thesis has focused on evaluating the activity of three gold(III) complexes with salicyl-TSC ligands against Toxoplasma gondii in vitro and in vivo. Among the compounds, one gold complex (C3) and its corresponding salicyl-TSC ligand (C4) demonstrated promising inhibition constants in vitro against the parasite (nanomolar range: C3 IC50 = 100 nM; C4 IC50 = 30 nM) and were selected for further analyses. Interestingly, after several days of in vitro treatment, T. gondii tachyzoites quickly adapted to these compounds, suggesting that the inhibitory effects of C3 and C4 were transient. To explore the mode of action of the drugs and the mechanisms behind the adaptation to the treatment, differential affinity chromatography coupled with mass spectrometry and proteomics (DAC-MS-proteomics) was employed. Initially, DAC-MS-proteomics was used to identify T. gondii tachyzoite proteins that interact with C3 and C4. Subsequently, DAC-MS-proteomics was carried out to compare the protein expression profiles of six T. gondii tachyzoite clones adapted to C3 and C4 treatment with those of non-adapted tachyzoites. DAC results suggested that C3 and C4 may interfere with protein biosynthesis and that adaptation to the treatment may be associated with the upregulated expression of tachyzoite transmembrane proteins and transporters, suggesting that the in vitro drug tolerance in T. gondii might be due to reversible, non-drug specific stress-responses mediated by phenotypic plasticity. Furthermore, the potential side effects of the compounds were investigated, especially regarding their impact on the immune system and embryonic development. Since neither compound exhibited any immunosuppressive effects on murine splenocytes, in vivo experiments were performed in a murine model of cerebral toxoplasmosis. However, the congenital murine model of toxoplasmosis was excluded from further studies due to the toxicity observed in the zebrafish embryo development model. In vivo efficacy of the compounds in the murine model of cerebral toxoplasmosis was evaluated by performing Real-time PCR on DNA extracted from the organs (brain, heart, and eyes) of mice after euthanasia. The analysis targeted the 529 bp gene of T. gondii, comparing parasite loads between infected, untreated mice and those treated with C3 and C4 compounds. The results from quantification of the parasite burden after in vivo treatments showed that neither compound was effective in reducing parasite burdens in the brain and eyes, while there is a statistically significant decrease of parasite burden in the heart after C4 treatment. This lack of efficacy in the brain and in the eyes is likely due to the inability of these compounds, like many other drugs tested for toxoplasmosis treatment, to cross the blood-brain barrier (BBB). Further studies should explore alternative routes of administration, which may enhance the compounds’ activity, and investigate their pharmacokinetics to assess how they are metabolized. This would help determine whether dosage adjustments are feasible. Another possibility could be testing these compounds in combination with standard therapies like sulfadiazine or pyrimethamine to potentially improve treatment outcomes. In summary, Toxoplasma gondii, the parasite responsible for toxoplasmosis, continues to cause significant challenges in treatment. Current therapies manage acute infection but have limitations, including incomplete parasite eradication and harmful side effects. Recent research focuses on developing new compounds, such as thiosemicarbazones and small molecules, which show promise in laboratory studies. These new drugs could offer better efficacy, reduced toxicity, and broader action, addressing the shortcomings of current treatments. This underlines the importance and continued necessity of studies like this to drive the discovery of more effective treatment options.