Sb-200, derivado 2-amino-tiofênico com potencial anti- Trypanosomatidae e imunomodulador

Abstract

Leishmaniasis and Chagas disease are among the most serious neglected tropical diseases (NTDs) and with the highest number of cases in the world. Currently, drugs available on the market have several problems, such as high cost, high toxicity, most are administered parenterally and many cases of parasite resistance. Therefore, the search for therapeutic alternatives is urgent. Thiophene is a five-membered compound that has sulfur as a heteroatom and two double bonds (at positions 2 and 4), forming an aromatic system. Thiophene derivatives occur in various organisms, such as fungi and plants, in addition to being easily synthesized in the laboratory, with numerous reports of antileishmania and anti-Trypanosoma cruzi activity. SB-200 is a 2-amino-thiophene derivative previously studied by our research group and which showed antileishmania activity against Leishmania (Leishmania) amazonensis. In light of previous promising results, the aim of this study was to evaluate the anti-Trypanosomatidae activity of SB-200 on other species of Leishmania and on the parasite Trypanosoma cruzi. The antileishmania activity of SB-200 was initially evaluated on promastigotes forms of Leishmania (Viannia) braziliensis, Leishmania (Leishmania) major and Leishmania (Leishmania) infantum, obtaining a mean inhibitory concentration (IC50) of 4.25 μM, 4, 65 μM and 3.96 μM, respectively. The anti-Trypanosoma cruzi activity of SB-200 was performed on epimastigotes and trypomastigotes, strain CL. IC50 values of 2.69 μM were obtained for epimastigotes while for trypomastigotes an IC50 of 29.69 μM was obtained. Due to a better result on the species L. infantum among all trypanosomatids, further studies on the amastigote form and mechanisms of action were carried out with this species. Cytotoxicity of the thiophene derivative was carried out in J774.A1 macrophages and VERO cells, obtaining an average cytotoxic concentration (CC50) of 42.52 μM and 39.2 μM, respectively. The selectivity index (SI) for L. infantum was 10.74 for macrophages and 9.89 for VERO cells. The antipromastigote activity of SB-200 is associated with damage to the plasma membrane, as the compound was able to increase permeability in an assay with SYTOX green, a DNA intercalator. Ultrastructural assays in L. infantum promastigotes using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed ruptures of the plasmatic membrane and revealed alterations such as an increase in cytoplasmic vacuoles, detachment of the nuclear membrane and fragmentation of the nucleus upon treatment with SB-200. In the investigation of the type of cell death promoted by the compound, an increase in the type of death by apoptosis was observed, with a secondary death by necrosis. Through the docking assay, SB-200 showed a molecular affinity with N-myristoyl transferase, important enzyme for the survival of the parasite. The antiamastigote activity of SB-200 was carried out on the L. infantum species, obtaining an EC50 of 2.85 μM and an IS of 14.97 in macrophages and 13.8 in VERO cells. The antimastigote activity of SB-200 is associated with an immunomodulatory activity, as it increased the levels of TNF-α, IL-12, IL-1β, nitric oxide (NO) and reactive oxygen species (ROS), while decreasing the levels of IL-10. The acute toxicity of SB-200 was investigated on Zophobas morio larvae, with an estimated lethal dose of >3000 mg/kg, proving the safety of the compound. Thus, it is concluded that the 2-amino-thiophene derivative SB-200 is a potential anti-Trypanosomatidae compound, and a candidate to advance in studies of new treatments for leishmaniasis and Chagas disease.