Atividade, seletividade e mecanismos de ação de diamidinas aromáticas e análogos sobre Trypanosoma cruzi: um enfoque sobre o kDNA

Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America, affecting more de 15 million people. Due to the limited efficacy and considerable toxicity, the therapy for Chagas disease is far from being considerable ideal and thus alternative drugs are urgently needed. In this context, with the collaborations of medicinal chemistry groups, our aim was to investigate the trypanocidal activity, selectivity, cellular targets and mechanisms of action of aromatic diamidines (ADs) and analogues. First, we tested the trypanocidal effect of eleven ADs upon intracellular amastigotes and trypomastigotes, their toxicity towards mammalian cells and their sub-cellular localization. Compounds 2, 5 and 7 were the most active, presenting IC50 values on the micromolar range and displaying high selectivity indexes for both T. cruzi forms. Using fluorescence microscopy (FM), all the compounds were localized in organelles rich in DNA, nucleus and mitochondrion (kDNA) and in bloodstream forms treated with the compounds 5 and 7 we observed ultrastructural damages on mitochondrial organelle, including kDNA disorganization. The accumulation of the diamidines was higher in the kDNA than in the nucleus, but such accumulation could not be correlated with a higher trypanocidal activity. Next, aiming to better understand the mechanism of action of diamidines and analogues, we investigated the possible correlation between kDNA binding properties of thirteen compounds with their anti-T. cruzi effect, through thermal denaturation (T m) and circular dichroism (CiD) studies. Our data demonstrated using the purified kDNA of epimastigotes or a conserved synthetic parasite sequence of 22-mer present in T. cruzi minicircles, that the strong interaction of the amidines with the kDNA is not a determinant factor to the triggering of the trypanocidal activity. Our next step was the evaluation of topological changes induced by ten aromatic compounds on T. cruzi kDNA, through gel mobility shifts and FM. The eletrophoretic studies were conducted by the incubation of kDNA fragments obtained by digestion with the endonucleases EcoRI e CvQI with the compounds and analysis by poliacrylamide gel. The diamidines DB889 and DB185 induced substantial mobility shifts in the fragmented bands. Additionally, by FM of whole kDNA network incubated with DB75 we observed the ability of this diamidine to cause a striking expansion of the kDNA network area. Taken together our results suggest that diamidines and related compounds provoke profound alterations in the normal topology of T. cruzi kDNA suggesting that this structure may represent one of the potential targets of these compounds. However, the compound preferential accumulation in kDNA, as well as their affinity and capability of inducing topological changes in that structure is not correlated with their anti-T. cruzi activity. These findings strongly suggest that other molecular mechanisms may be also operating primarily or secondarily to the drug:KDNA interaction. Further studies are needed to better identify the mechanisms involved in the activity of this class of chemical, aiming to contribute to the rational design of lead compounds for the treatment of Chagas disease .