Antonio Morello

Platinum-based complexes of bioactive 3-(5-nitrofuryl)acroleine thiosemicarbazones showing anti-Trypanosoma cruzi activity.

Eight new platinum(II) complexes with 3-(5-nitrofuryl)acroleine thiosemicarbazones showing anti-trypanosomal activity were synthesized, characterized and in vitro evaluated. Most of the complexes showed IC(50) values in the micromolar range against two different strains of Trypanosoma cruzi, causative agent of Chagas disease (American Trypanosomiasis). In addition, most of the newly developed complexes, together with the analogous platinum 5-nitrofuraldehyde containing thiosemicarbazones previously reported, resulted more active than the reference trypanocidal drug nifurtimox on the infective trypomastigote form of the parasite. Their capacity to produce free radicals that could lead to parasite death was evaluated by ESR experiments in the parasite and by respiration measurements. Compounds were tested for their DNA interaction ability. Results showed that some of the compounds could act as dual inhibitors in the parasite, through production of toxic free radicals and interaction with DNA. All the results were compared with those previously reported for the free ligands, the analogous palladium(II) compounds and the previously reported series of platinum(II) compounds.

Buthionine Sulfoximine Increases the Toxicity of Nifurtimox and Benznidazole to Trypanosoma cruzi

ABSTRACT l-Buthionine (S,R)-sulfoximine (BSO) increased the toxicity of nifurtimox and benznidazole toward the epimastigote, trypomastigote, and amastigote forms of Trypanosoma cruzi. BSO at 500 μM decreased total glutathione-derived thiols by 70 to 80% in 48 h. In epimastigotes, 500 μM BSO decreased the concentration of nifurtimox needed to inhibit constant growth of the parasites by 50%, from 14.0 to 9.0 μM, and decreased that of benznidazole from 43.6 to 24.1 μM. The survival of epimastigotes or trypomastigotes treated with nifurtimox or benznidazole, as measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction, was significantly decreased by 500 μM BSO. In Vero cells infected with amastigotes, 25 μM BSO was able to potentiate the effect of nifurtimox and benznidazole as measured by the percentage of infected Vero cells multiplied by the average number of intracellular amastigotes (endocytic index). At 0.5 μM nifurtimox, the proportion of Vero cells infected decreased from 27 to 20% and the endocytic index decreased from 2,500 to 980 when 25 μM BSO was added. Similar results were obtained with benznidazole- and BSO-benznidazole-treated cells. This study indicates that potentiation of nifurtimox or benznidazole by BSO could decrease the clinical dose of both drugs and diminish the side effects or the length of therapy.

Natural and synthetic naphthoquinones active against Trypanosoma cruzi: an initial step towards new drugs for Chagas disease.

Chagas disease is one of the most important endemic diseases in Latin America, caused by Trypanosoma cruzi. The drugs used for the treatment of this disease, nifurtimox and benznidazole, are toxic and present severe side effects. The need of effective drugs, without adverse effects, has stimulated the search for new compounds with potential clinical utility. An overview of a number of natural naphthoquinones tested against T. cruzi parasites is provided. Among natural naphthoquinones, lapachol, β-lapachone and its α-isomer have demonstrated useful trypanocidal activities. In the search for new trypanocidal agents, this review outlines different structural modifications of natural quinones, as well as synthetic quinones, which have been subjected to trypanocidal studies. This review summarizes the mechanism of action and structure-activity relationships of the quinone derivatives, including some theoretical calculations that discuss the correlation of stereo electronic properties with the trypanocidal activity. In this context, this review will be useful for the development of new antichagasic drugs based mainly on structural modification of natural quinones.

Chagas disease: Present status of pathogenic mechanisms and chemotherapy

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20%-30% of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the hosts immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity. In addition, there is increasing evidence that cyclooxygenase inhibitors present an important effect on T. cruzi infection. Therefore, we found that aspirin reduced the intracellular infection in RAW 264.7 cells and, decreased myocarditis extension and mortality rates in mice. However, the long-term benefit of prostaglandin inhibition for Chagasic patients is still unknown.

ESR Spin Trapping Studies of Free Radicals Generated from Nitrofuran Derivative Analogues of Nifurtimox by Electrochemical and Trypanosoma cruzi Reduction

Electron spin resonance (ESR) spectra of radicals obtained from two analogues of the antiprotozoal drug nifurtimox by electrolytic and Trypanosoma cruzi reduction were analyzed. The electrochemistry of these compounds was studied using cyclic voltammetry. STO 3-21G ab initio and INDO molecular orbital calculations were performed to obtain the optimized geometries and spin distribution, respectively. The antioxidant effect of glutathione on the nitroheterocycle radical was evaluated. DMPO spin trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. Nitro1 and Nitro2 nitrofuran analogues showed better antiparasitic activity than nifurtimox. Nitro2 produced oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of either the hydroxyl radical or the Nitro2 analogue radicals. These results are in agreement with the biological observation that Nitro2 showed anti-Chagas activity by an oxidative stress mechanism.

Glutathione and trypanothione in several strains of Trypanosoma cruzi: effect of drugs.

Glutathione (GSH), trypanothione (T(SH)2) and glutathionyl spermidine (GSH-SP) concentrations were determined in the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The concentrations of GSH, T(SH)2 and GSH-SP, expressed as nmol of GSH per g of parasite fresh weight, were 60.1, 397.8 and 103.9, respectively, for the Tulahuén strain. For the DM 28c clone, the values were 113.9, 677.9 and 164.1, respectively, and for the LQ strain they were 199.1, 1100.5 and 55.3, respectively. When the parasites were treated with 10 microM nifurtimox or 50 microM benznidazole for 2 h, the concentrations of all three reduced thiols decreased strongly. The total amount of T(SH)2 decreased by more than 50%. Treatment of the parasites with 5 mM buthionine sulfoximine, an inhibitor of GSH synthesis, for 6 h diminished the concentrations of the reduced thiols by between 27% and 53% with respect to the controls. Cyclohexylamine, an inhibitor of spermidine synthesis, decreased the concentrations of T(SH)2 and GSH-SP but not that of GSH. It is possible to conclude from this study that trypanothione is the most important thiol involved in the detoxication of nifurtimox and benznidazole in T. cruzi and that electrophilic reduced metabolites of both drugs are most probably conjugated with GSH, T(SH)2 and GSH-SP, thus decreasing their concentrations. GSH biosynthesis is an important drug target.

Antiproliferative and Uncoupling Effects of Delocalized, Lipophilic, Cationic Gallic Acid Derivatives on Cancer Cell Lines. Validation in Vivo in Singenic Mice

Tumor cells principally exhibit increased mitochondrial transmembrane potential (ΔΨ(m)) and altered metabolic pathways. The therapeutic targeting and delivery of anticancer drugs to the mitochondria might improve treatment efficacy. Gallic acid exhibits a variety of biological activities, and its ester derivatives can induce mitochondrial dysfunction. Four alkyl gallate triphenylphosphonium lipophilic cations were synthesized, each differing in the size of the linker chain at the cationic moiety. These derivatives were selectively cytotoxic toward tumor cells. The better compound (TPP(+)C10) contained 10 carbon atoms within the linker chain and exhibited an IC50 value of approximately 0.4-1.6 μM for tumor cells and a selectivity index of approximately 17-fold for tumor compared with normal cells. Consequently, its antiproliferative effect was also assessed in vivo. The oxygen consumption rate and NAD(P)H oxidation levels increased in the tumor cell lines (uncoupling effect), resulting in a ΔΨ(m) decrease and a consequent decrease in intracellular ATP levels. Moreover, TPP(+)C10 significantly inhibited the growth of TA3/Ha tumors in mice. According to these results, the antineoplastic activity and safety of TPP(+)C10 warrant further comprehensive evaluation.

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor: Effects on Trypanosoma cruzi epimastigotes

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.t‐Butyl‐4‐hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.

Involvement of thiol metabolism in resistance to glucantime in Leishmania tropica

Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.

Effects and mode of action of 1,4-naphthoquinones isolated from Calceolaria sessilis on tumoral cells and Trypanosoma parasites

The naphthoquinones 2-hydroxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-1), (-)-2,3,3-trimethyl-2-3-dihydronaphtho[2,3-b]furan-4,9-quinone (CS-3), and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-5) isolated from Calceolaria sessilis were tested against Trypanosoma cruzi epimastigotes, the TA3 tumor cell line and the methotrexate-resistant subline TA3-MTX-R. Naphthoquinone CS-3 was the most active; the 50% culture growth inhibition (I50) on T. cruzi (Tulahuén and LQ strain and DM28c clone) was at concentrations ranging from 2.1 to 5.2 mumolar. Also CS-3 inhibited TA3 and TA3-MTX-R culture growth with an I50 of 2.1 and 3.8 mumolar, respectively. Naphthoquinone CS-3 inhibited the respiration of the tumor cells by interfering with the electron transport at some point between NADH and ubiquinone. The respiration of T. cruzi was not inhibited by naphthoquinone CS-3. Naphthoquinone CS-3 produced a temporary increase of oxygen consumption in T. cruzi and tumor cells, suggesting the generation and participation of free radicals.