Mario Faúndez

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.

Buthionine Sulfoximine Has Anti-Trypanosoma cruzi Activity in a Murine Model of Acute Chagas' Disease and Enhances the Efficacy of Nifurtimox

ABSTRACT l-Buthionine (S,R)-sulfoximine (BSO) at a dose of 220 mg/kg of body weight/day showed an anti-Trypanosoma cruzi effect in infected mice, increasing their survival rate and decreasing the parasitemias and parasite burden in the hearts. Treatment with BSO plus nifurtimox caused an increase in the survival rate in comparison to the rates with treatment with each drug alone.

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole.

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor beta and PGE(2) activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE(2) synthesis inhibition. Nevertheless, NO() levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.

Mechanisms underlying the inhibition of the cytochrome P450 system by copper ions.

Copper toxicity has been associated to the capacity of free copper ions to catalyze the production of superoxide anion and hydroxyl radical, reactive species that modify the structure and/or function of biomolecules. In addition, nonspecific Cu2+‐binding to thiol enzymes, which modifies their catalytic activities, has been reported. Cytochrome P450 (CYP450) monooxygenase is a thiol protein that binds substrates in the first and limiting step of CYP450 system catalytic cycle, necessary for the metabolism of lipophilic xenobiotics. Therefore, copper ions have the potential to oxidize and bind to cysteinyl residues of this monooxygenase, altering the CYP450 system activity. To test this postulate, we studied the effect of Cu2+ alone and Cu2+/ascorbate in rat liver microsomes, to independently evaluate its nonspecific binding and its pro‐oxidant effects, respectively. We assessed these effects on the absorbance spectrum of the monooxygenase, as a measure of structural damage, and p‐nitroanisole O‐demethylating activity of CYP450 system, as a marker of functional impairment. Data obtained indicate that Cu2+ could both oxidize and bind to some amino acid residues of the CYP450 monooxygenase but not to its heme group. The differences observed between the effects of Cu2+ and Cu2+/ascorbate show that both mechanisms are involved in the catalytic activity inhibition of CYP450 system by copper ions. The significance of these findings on the pharmacokinetics and pharmacodynamics of drugs is discussed. Copyright

Melatonin protects the cytochrome P450 system through a novel antioxidant mechanism

Melatonin, an endogenous hormone, is used as an antioxidant drug in doses quite higher than the endogenous circulating levels of this hormone. Hepatic endoplasmic reticulum contains the cytochrome P450 (CYP450) system, which catalyzes one biotransformation pathway of melatonin; this organelle is also one of the main sources of reactive oxygen species in cells. Therefore, we proposed that the antioxidant activity of this hormone may have a biological relevance in the organelle where it is biotransformed. To evaluate this postulate, we used Fe(3+)/ascorbate, an oxygen free radical generating system that leads to lipid peroxidation, loss of protein-thiol content, and activation of UDP-glucuronyltransferase in rat liver microsomes. We found that mM concentrations of melatonin prevented all these oxidative phenomena. We also found that Fe(3+)/ascorbate leads to structural alterations in the CYP450 monooxygenase, the enzyme that binds the substrate in the CYP450 system catalytic cycle, probably through direct oxidation of the protein, and also inhibited p-nitroanisole O-demethylation, a reaction catalyzed by the CYP450 system. Notably, melatonin prevented both phenomena at microM concentrations. We provide evidence suggesting that melatonin may be oxidized by oxygen free radicals. Thus, we postulate that melatonin may be acting as an oxygen free radical scavenger, and Fe(3+)/ascorbate-modified melatonin would be directly protecting the CYP450 system through an additional specific mechanism. Pharmacological relevance of this phenomenon is discussed.

Synthesis and Pharmacophore Modelling of 2,6,9-Trisubstituted Purine Derivatives and Their Potential Role as Apoptosis-Inducing Agents in Cancer Cell Lines

A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. Twelve compounds were obtained by a three step synthetic procedure using microwave irradiation in a pivotal step. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method and flow cytometry analysis on four cancer cells lines and Vero cells. Three out of twelve compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide, in three out of four cancer cell lines assayed with considerable selectivity. Preliminary flow cytometry data suggests that compounds mentioned above induce apoptosis on these cells. The main structural requirements for their activity for each cancer cell line were characterized with a preliminary pharmacophore model, which identified aromatic centers, hydrogen acceptor/donor center and a hydrophobic area. These features were consistent with the cytotoxic activity of the assayed compounds.

Pyrogallol red oxidation induced by superoxide radicals: application to evaluate redox cycling of nitro compounds.

The bleaching of the pyrogallol red (PGR) dye mediated by superoxide anion radicals (O(2)(-)) generated from the xanthine/xanthine oxidase system (X/XO) was studied by UV-visible spectrophotometry. The absorption band (at 540 nm) of PGR quickly decreased in the presence of X/XO, implying an efficient reaction of O(2)(-) with PGR. The process was unaffected by catalase (CAT), but completely abolished by superoxide dismutase (SOD). A mechanism of the reaction involving the consumption of one PGR molecule by two O(2)(-) to generate one molecule of H(2)O(2) is proposed. PGR was used as a probe to estimate the rate of O(2)(-) generation in redox cycling reactions of a series of nitro compounds mediated by rat liver microsomes. The consumption of PGR induced by the redox cycling of nitrofurantoin was totally eliminated by the addition of SOD but unaffected by CAT. The initial rate of consumption of PGR mediated by the redox cycling of others nitro derivatives follows the order: furazolidindione > nitrofurantoin > nifurtimox > benznidazole > chloramphenicol. We concluded that PGR can be used as a probe to estimate the release of O(2)(-) from enzymatic systems or from the redox cycling of nitro compounds.

Combined CoMFA and CoMSIA 3D-QSAR study of benzimidazole and benzothiophene derivatives with selective affinity for the CB2 cannabinoid receptor

ABSTRACT The preceding years have brought an exponential increase in our understanding of the endocannabinoid system (ECS), including the knowledge of CB1 and CB2 cannabinoid receptors, endocannabinoids, and the enzymes that synthesize and degrade endocannabinoids. Among these ECS components CB2 receptors have been the subject of considerable attention, primarily due to their promising therapeutic potential to treat numerous pathologies while avoiding the adverse psychotropic effects that can accompany CB1 receptor–based therapies. Recently, our research group has reported a new series of non‐cytotoxic benzo[d]imidazoles and benzo[b]thiophenes displaying high CB2/CB1 selectivity index. In order to investigate the structural requirements for CB2 ligands and to derive a predictive model that can be used for the design of novel selective CB2 ligands, a three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) study was performed on the above mentioned chemical series employing comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) techniques. The CoMFA and CoMSIA models displayed high external predictability (rpred2 0.919 and 0.908) and good statistical robustness. Valuable information regarding the steric, electrostatic and hydrophobic properties of the molecules was obtained, and several modifications around both heterocycles were evaluated with the aim to generate new promising series of benzo[d]imidazoles and benzo[b]thiophenes derivatives displaying high CB2 selectivity and low toxicity. Graphical abstract Figure. No Caption available.

New imidoyl-indazole platinum (II) complexes as potential anticancer agents: Synthesis, evaluation of cytotoxicity, cell death and experimental-theoretical DNA interaction studies

Four new neutral N,N imidoyl-indazole ligands (L1, L3, L6, L7) and six new Pt(II)-based complexes (C1-5 and C7) were synthesized and characterized by spectroscopic and spectrometric techniques. Additionally, compounds L6, L7, C3, C5 and C7 were analyzed using X-ray diffraction. An evaluation of cytotoxicity and cell death in vitro for both ligands and complexes was performed by colorimetric assay and flow cytometry, in four cancer cell lines and VERO cells as the control, respectively. Cytotoxicity and selectivity demonstrated by each compound were dependent on the cancer cell line assayed. IC50 values of complexes C1-5 and C7 were lower than those exhibited for the reference drug cisplatin, and selectivity of these complexes was in general terms greater than cisplatin on three cancer cell lines studied. In HL60 cells, complexes C1 and C5 exhibited the lowest values of IC50 and were almost five times more selective than cisplatin. Flow cytometry results suggest that each complex predominantly induced necrosis, and its variant necroptosis, instead of apoptosis in all cancer cell lines studied. DNA binding assays, using agarose gel electrophoresis and UV-visible spectrophotometry studies, displayed a strong interaction only between C4 and DNA. In fact, theoretical calculations showed that C4-DNA binding complex was the most thermodynamic favorable interaction among the complexes in study. Overall, induction of cell death by dependent and independent-DNA-metal compound interactions were possible using imidoyl-indazole Pt(II) complexes as anticancer agents.