Amidine derivatives and Leishmania amazonensis: an evaluation of the effect of nitric oxide (NO) production on the parasite-macrophage interaction.

Previous work has demonstrated that N-N'-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes and Trypanosoma evansi trypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infection's supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production by L. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present in Leishmania parasites for leishmanicidal drugs.

Protein kinase A of Leishmania amazonensis as a potential target for methoxy-amidine.

Cyclic 3',5'-adenosine monophosphate (cAMP) is one of the most important signaling molecules for cell growth and differentiation in several systems including protozoal parasites such as Trypanosoma cruzi and Leishmania species. The most important event during Leishmania developmental cycle is the differentiation of procyclic into metacyclic promastigotes, which is associated with the appearance of pathogenicity. As previously demonstrated Leishmania amazonensis metacyclogenesis is associated with an increase of a protein kinase A activity, and therefore further studies on the activity of this phosphorylating enzyme as a target for chemotherapy were performed. Among several amidine derivatives tested by the authors against trypanosomatids (T. cruzi, T. evansi and L. amazonensis) the most effective compounds was defined as that with a methoxy group as substituent. In this work the inhibitory effect of this derivative on the phosphorylating activity of cAMP-dependent protein kinase (PKA) of promastigotes (containing high amounts of metacyclic forms) and axenic amastigotes of L. amazonensis is demonstrated. Soluble fractions (SF) and enriched membrane fractions (MF) were submitted to anion exchange chromatography in a DEAE-cellulose column and the collected fractions used to evaluate the phosphorylating activity associated with cAMP, in the presence/absence of methoxy-amidine and pentamidine (CAS 100-33-4), the latter being used as reference drug.

Effect of amidine derivatives on Leishmania amazonensis axenic amastigotes. Preliminary studies of structure-activity relationships

In a previous work, searching for new drugs with high efficiency and low toxicity, the in vitro antileishmanial activity of a series of seven amidine derivatives against Leishmania amazonensis promastigotes was reported. In order to compare the potential difference in the susceptibility between the two developmental stages of the parasite to these compounds, an analysis of the effects of these amidine derivatives on promastigotes and axenically grown amastigotes of L. amazonensis was carried out. Among the N,N'-diphenyl-4-R-benzamidine (where R = H, Cl, Br, CN, NO2, CH3 and OCH3) derivatives studied, the most active compounds in both developmental stages were those with -Br, -Cl, -NO2, and -OCH3 substituents, the nonsubstituted ones being the least active. However, the susceptibility to the drugs varied in these forms, being higher in promastigotes. The electronic theoretical parameters calculated by molecular modelling using the semi-empirical AM1 method showed good correlation with substituent constant (sigma para, sigma 1 and F). The chemical structure and biological activity relationships were investigated in all compounds, although a small but significant correlation was observed. The obtained results suggest that other factors than the electronic parameters have an influence on the biological response.

Leishmania amazonensis: long-term cultivation of axenic amastigotes is associated to metacyclogenesis of promastigotes.

Axenic Leishmania amazonensis cultures were obtained (with 2 years of follow-up) in Schneider's medium at pH 5.5 and a temperature of 32 degrees C. Differentiation of promastigotes into amastigotes is directly associated with the metacyclogenesis rate of the original promastigotes. Promastigote cultures containing different percentages of metacyclic forms, as evaluated by the complement lysis test, resulted in axenic amastigote cultures containing a variable amount of undifferentiated forms. We observed that a culture originally containing a large amount of metacyclic forms (73%) underwent a complete differentiation process, resulting in a 100% amastigote culture with no promastigote forms. All other cultures (with a decreasing amount of metacyclic forms: 50% and fewer) failed to differentiate completely, resulting in cultures with increasing percentages of promastigote contamination. The amastigote/promastigote rate in these cultures has been maintained at the same level thus far. The axenic Leishmania amazonensis amastigotes were cryopreserved, thawed as such, and successfully cultivated under appropriately defined growth conditions.