Application of supramolecular chemistry in the search for active compounds against trypanosoma cruzi infection in mouse model/aplicación de la química supramolecular en la búsqueda de compuestos activos frente a la infección por trypanosoma cruzi en modelo Murino

Resumen

According to the World Health Organization (WHO) Chagas disease or American trypanosomiasis is a parasitic, systemic, chronic disease caused by the protozoan Trypanosoma cruzi. It is spread naturally by triatomine insects, especially the so-called "kissing bugs" that normally colonize poor-quality housing. It is also transmitted by transfusions, congenitally, via organ transplants and through contaminated food. Chagas disease is endemic in 21 countries in the Americas, where it is estimated that about 100 million people are at risk of infection, with around 8 million infected, and a total of 56,000 new cases annually including all forms of transmission, leading to 12,000 deaths per year. Current treatments, benznidazole and nifurtimox, exhibit limited effectiveness, only being curative during the acute phase of infection and not in the chronic phase, which is when most cases are diagnosed. Furthermore, administration involves a series of high-toxicity side effects. In addition, there is no vaccine against this parasitic infection. For several years we have been collaborating with various chemical synthesis groups around Spain, which has resulted in the production of a series of joint publications, as well as national and international collaborative projects. This cooperative work has allowed us to gain a wide experience and knowledge of a number of chemical families which display anti-protozoal activity against Trypanosoma cruzi and Leishmania sp. In this study, we looked at five specific families with different chemical natures to observe their in vitro and in vivo activity against T. cruzi. These were: N, N'-Squaramides (Department of Chemistry, Faculty of Science, University of the Balearic Islands, Palma de Mallorca), Phthalazines (Department of Organic Chemistry, Faculty of Chemistry, Complutense University, Madrid / Institute of Medical chemistry, Organic Chemistry Center M. Lora-Tamayo, CSIC), Abietic Acid Derivatives (Department of Organic chemistry, University of Granada), Tetradentated polyamines (QBIS Research Group, Institute of Computational Chemistry and Catalysis (IQCC) and the Department of Chemistry, University of Girona), and Scorpiand-like azamacrocycles (Institute of Molecular Science, Department of Inorganic Chemistry, University of Valencia / Department of Organic Chemistry, University of Valencia). The experimental biology was conducted entirely in the Department of Parasitology, Faculty of Sciences, University of Granada, with the exception of the "Trypanothiona reductase (TR) inhibition assay" and the "in vivo bioluminescence imaging study", which were developed in the Biochemie-Zentrum der Universität Heidelberg (Germany) and the London School of Hygiene and Tropical Medicine (UK) respectively, during two pre-doctoral stays. The entire study was carried out through a series of experiments that can be classified into three phases: the in vitro stage, in vivo stage, and a study of the mechanism of action. During the first two stages, compounds were screened: those with elevated activity were selected and those that did not meet minimum requirements of effectiveness were discarded. During the first stage of in vitro screening, the compounds were tested at different concentrations against different forms of the parasite and mammalian cells (cytotoxicity); dividing parasite IC50 between mammalian cells IC50 (the concentration at which 50% growth inhibition occurs) a specific selectivity index (SI) for each compound was obtained. This SI was indicative of the specificity toxicity of that compound had against parasites versus mammalian cells. Based on this, the compounds exhibiting the best selectivity indices according to the criteria established in the literature were selected for the next stage. The next step was to test these compounds selected from the in vitro phase in an experimental animal model, the Balb/c murine model. The mice were infected and once the infection had been confirmed the treatment was started. This treatment was continued for 5 days (acute phase). Throughout the acute phase (up to 40 days post-infection), the mice were bled every three days to follow the parasitemia levels, and blood samples were obtained for serum as well as to assess the levels of antibodies, and identify common biochemical parameters in the serology that could reveal signs of toxicity associated with the compounds. The mice were kept in the same conditions for at least 120 days, i.e., the time period of the chronic phase of infection characterized by a decrease in blood parasitemia that reaches an almost undetectable level. To determine the degree of effectiveness/cure of the therapy administered, an immunosuppressive treatment was applied to the mice, to induce reactivation of parasitema in cases where there was no cure. Similarly, the specific target organ for this strain of parasite, the heart, was analyzed post mortem by PCR to determine the presence or absence of the parasite. The final stage of this study included a search for possible mechanisms of action of those compounds that had been effective in the in vivo treatment. For this, tests were performed at three levels: energy metabolism, by analyzing the variation in metabolite excretion to the culture medium by 1H-NMR; ultrastructural analysis, by transmission electron microscopy; and antioxidant defense, by in vitro inhibition studies of the parasite-specific enzymes iron superoxide dismutase and trypanothione reductase. Finally, the possible synergistic actions of these selected compounds were studied using in vitro drug-combination assays and later in vivo studies of combinations with very similar working protocols to those in a clinical situation. The results obtained are summarized in several conclusions about the potential use of these compounds as trypanocidal agents, areas for improvement in this line of research, and what future guidelines could be implemented to take an immediate step further in the preclinical phase. Also, it should be noted that many of these families of compounds are patent-protected and there is a commitment to continued improvements in their pharmaco-kinetic properties and working protocols in order to achieve better results.