Análise de novos protótipos leishmanicidas : inibição seletiva de topoisomerase de Leishmania sp. por substâncias naturais e semissintéticas

Abstract

Leishmaniasis is one of the most important neglected tropical diseases in terms of drug discovery and development. Furthermore, the chemotherapy used to treat this disease has been proved to be highly toxic. The aim of this work was to evaluate the leishmanicidal effect of propolis derivatives collected in the semi-arid region of Bahia and a series of natural and semisynthetic alkamides, as well to evaluate the action of these on the enzymatic activity of Leishmania and human DNA topoisomerase. The chemical study of propolis was carried by high resolution mass spectrometry analysis; and alkamides were obtained from the acid chloride preparation reactions, the preparation of the amides from the acid chloride and the amine reactions, and the aminolysis of the phenylethylamine with the methyl p-hydroxycinnamate reactions. The cytotoxic effects were assessed on macrophages J774 by MTT assays. The inhibitory effect, in vitro assays, on promastigote and amastigote forms of L. amazonensis and L. chagasi were evaluated by MTT assay and cell J774 infected with promastigotes forms of these species assay, respectively. The inhibitory effect on promastigote and amastigote forms of L. infantum iFRP70 was assessed by the capacity of infrared light emission by strains iFRP70. The inhibition of topoisomerase activity was assessed by DNA supercoiled relaxation assay. Cleavage complex formation was assessed by the capacity to maintain the cleavage in the DNA strand over 30 minutes. The ethanolic extract, the hexane, ethyl acetate and methanolic fractions of propolis, and flavonoids naringenin and isoramnetin, did not present deleterious effects on the J774 cells until to the maximum concentration tested (100 μM). These flavonoids affected the enzymatic activity of leishmania topoisomerase, interfering in the enzyme's capacity to relax the DNA supercoiled and did not affected the enzymatic activity of humana topoisomerase. The alkamides tested, RAC4, RAC9, RAC9, RAC12 and RAC16 showed a significant effect against promastigotes and amastigotes forms of L. amazonensis, L. chagasi and L. infantum iFRP70, with a 50% inhibitory concentration (IC50) below 10 μM and maximum effect above 60%. These alkamides tested at concentrations ranging from 100 to 3.15 μM, inhibited the capacity of leishmania topoisomerase to DNA supercoiled significantly interfering in the enzymatic activity, especially RAC8 that was able to inhibit activity from 6.25 μM, lower inhibitory concentration observed. These alkamides did not inhibit the enzymatic activity of human topoisomerase. When evaluated the formation of cleavage complex, the RAC4, RAC8, RAC9, RAC12 and RAC16 were able to maintain the stability of the cleavage complex. Thus, this work demonstrated that the natural and semi-synthetic substances had a leishmanicidal effect against L. amazonensis, L. chagasi and L. infantum iFRP70, as well as selectively inhibited the catalytic activity of the enzyme DNA topoisomerase of leishmania, to be used as a prototype for the development of new leishmanicidal drugs.