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We have evaluated the cytotoxic properties against the protozoan Leishmania infantum of four water soluble cationic trans-Pt(II)Cl(2) compounds containing as inert groups NH3 and piperazine (1), 4-picoline and piperazine (2), n-butylamine and piperazine (3), and NH3 and 4-piperidino-piperidine (4). The leishmanicidal activity of compounds 3 and 4 against promastigotes of the parasite Leishmania infantum was 2.5- and 1.6-times higher than that of the cytotoxic drug cis-diamminedichloroplatinum(II), respectively. Interestingly, compounds 3 and 4 produce in Leishmania infantum promastigotes a higher amount of programmed cell death than cisplatin, which is associated with cell cycle arrest in G2/M. In contrast to cis-diamminedichloroplatinum(II), binding of compounds 3 and 4 to calf thymus DNA induces conformational changes more similar to those of trans-diamminedichloroplatinum(II) that may be attributed to denaturation of the double helix. Similarly to cis-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II), the interaction of compounds 3 and 4 with ubiquitin results in an increase of the alpha-helix content of the protein as observed by circular dichroism spectroscopy. However, fluorescence studies indicate that compounds 3 and 4 produce a decrease in the fluorescence of the tyrosine 59 residue of ubiquitin higher than both cis-diamminedichloroplatinum(II) and trans-diamminedichloroplatinum(II). Altogether, our results suggest that the biochemical mechanism of cytotoxic activity of compounds 3 and 4 against Leishmania infantum must be different from that of cis-diamminedichloroplatinum(II). To the best of our knowledge, compounds 3 and 4 are the first reported trans-platinum complexes that show antiparasitic activity.

Total-reflection X-ray fluorescence has been used to study whether the Leishmania infantum kinetoplastid membrane protein-11 is a Ca2+-binding protein. The 108 amino acid helix-loop-helix protein has the loop region located between residues 45 and 57, having similarity to the EF-hand motifs. In particular, the sequence alignment of the putative motif revealed the existence of 67% similarity and 33% identity with the EF-hand of the plasmodia-specific 40-kDa protein from Physarum polycephalum. To address the type of conformational changes induced by Ca2+ binding, circular dichroism and fluorescence spectroscopy were used. The data showed that Ca2+ induces changes in both the secondary and tertiary structure of the protein in a temperature- and pH-dependent way. It also induces the precipitation of the protein at pH 7.5, in contrast with what occurs at pH 5.0, and the precipitation process can be reverted by addition of EGTA. At acidic pH values the complex EGTA-Ca2+ causes drastic structural changes, forcing the protein to adopt a structure close to that of a random coil. Because, at acidic pH values, protein:Ca2+:EGTA ternary complexes may be formed, the drastic change may be attributed to the presence of a high density of EGTA negative charges in the neighborhood of the alpha-helices.

Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell surface of kinetoplastids, and acts as a potent B- and T-cell immunogen during Leishmania infection. Here we report that the Leishmania infantum KMP-11 secondary structure adopts mainly an alpha-helical conformation at pH 7.5 and that its urea- and thermally-induced unfolding constitute a fully reversible two-step process. This allows estimation of a half-denaturation temperature of approximately 65 degrees C, a delta GDH2O at 20 degrees C of approximately 14.63 kJ.mol-1, and an increment of the reaction heat of approximately 183.92 kJ.mol-1 and an entropy of approximately 543.4 J.mol-1.deg-1, respectively, for the native-denatured equilibrium of the KMP-11 in solution. We also report that the KPM-11 protein is induced to adopt a molten globule state at a pH range between pH 4 and pH 6. As a whole, the stability and the specific features of the denaturing effect induced by changes in pH are similar in KMP-11 to various other lipoproteins.