Uso racional de inibidores de fosfodiesterases como abordagem terapêutica para Doença de Chagas

Discovered in 1909 by the Brazilian physician and researcher Carlos Chagas, Chagas disease (DC) is part of the group of twenty pathologies known as Neglected Tropical Diseases, and is caused by the hemoflagellate protozoan Trypanosoma cruzi. The reference drugs benznidazole (BZ) and nifurtimox have important effects, such as considerable side effects, limited efficacy in the chronic phase and strains naturally resistant to both. In this context, research groups, including the PDE4NPD consortium, have been working on the identification, optimization and screening of phosphodiesterase inhibitors (PDEs) from commercial libraries focused on human PDEs and based on structural amd functional differences of parasitic isoforms. Even with a high degree of conservation, these enzymes, which are present as five isoforms in trypanosomatids and have as exclusive function the hydrolysis of cyclic nucleotides cAMP and cGMP, important intracellular messengers that interfere with osmoregulation, differentiation, infectivity and parasite survival, which turns them in interesting for new therapeutic approaches. Thus, the aim of this research was to perform a phenotypic screening of PDE inhibitors on T. cruzi through in vitro and in vivo studies. This work resulted in seven articles, in which we approached different aspects of 66 compounds distributed in three chemical classes: pyrazolones, phthalazinones and imidazoles In the in vitro studies, we considered different strains and DTUs (Discrete Typing Units), an anti-T. cruzi activity, toxicity and selectivity towards mammalian cells, compound-induced morphological changes, potential effect on host cell physiology, impact of compound combination therapy with BZ, as well as target validation measured by cAMP levels in parasites treated (or not) with inhibitors and sequencing of parasite isoforms (Y and Colombiana strains). We observed that, except for imidazoles, most inhibitors show greater activity on intracellular forms than blood trypomastigotes. We ruled out a possible action on the microbicidal potential of the host cell through pre-infection effect evaluation, which did not result in parasitic load reduction, and free amastigote analysis, revealing the maintenance of the same activity range when compared to intracellular ones. It suggests several hypotheses: metabolic differences between the two forms, whose biometabolization is more intense in the proliferative forms in relation to trypomastigotes, and as currently shown unprecedentedly, amastigotes exhibiting superior efflux of cAMP compared to bloodstream forms, besides some differences regarding expression of PDE isoforms. These factors may result in different susceptibility profiles for most compounds, being more potent over amastigotes However, although they do not lead to the lyses of trypomastigotes, the compounds profoundly induce morphological changes in parasites, impacting their infectious potential. The most promising candidate, the pyrazolone NPD-227, was conducted to in vivo acute toxicity trials and to approach murine models. We confirmed the low toxicity of this inhibitor with NOAEL (non-observed adverse effects level) of 400 mpk. Its action on acute experimental T. cruzi infection, in monotherapy regimens and under co-administration with BZ, revealed a superior effect of combo approach when compared to monotherapy, both in parasitemia and animal survival levels. Human PDE isoforms enjoy a long history as pharmacological targets for a broad spectrum of clinical conditions and this reallocation of know-how and technology offers new opportunities for identifying therapeutic candidates for a CD