3-H-[1,2]Dithiole as a New Anti-Trypanosoma cruzi Chemotype: Biological and Mechanism of Action Studies.

The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.

The pharmacological inhibition of sterol biosynthesis in Leishmania is counteracted by enhancement of LDL endocytosis.

Leishmania parasites, despite being able to synthesize their own sterols, acquire and accumulate significant amounts of cholesterol through low density lipoprotein (LDL) particle endocytosis. The role of this system in Leishmania amazonensis promastigotes under pharmacological pressure by sterol biosynthesis inhibitors (SBIs) was investigated. First, thin layer chromatography demonstrated that L. amazonensis promastigotes, in response to ergosterol biosynthesis inhibition by treatment with 4.0 and 6.0 µM ketoconazole or miconazole, accumulate up to two times more cholesterol than controls. The treatment of promastigotes with ketoconazole and simvastatin, two SBIs with non-related mechanisms of action, showed that both drugs induce increases in (125)I-LDL endocytosis in a dose-dependent manner, indicating that the accumulation of exogenous cholesterol is due to the enhancement of LDL uptake. Finally, it was demonstrated that L. amazonensis promastigotes were rendered more susceptible to treatment with SBIs (ketoconazole, miconazole, simvastatin and terbinafine) in the absence of exogenous cholesterol sources, with a reduction of the IC50s of about 50% in three of the four tested drugs. These results show that the exogenous cholesterol uptake system in L. amazonensis plays a role as a compensatory mechanism in response to the presence of SBIs, suggesting that it may be a potential pharmacological target.

Trypanosoma cruzi targets for new chemotherapeutic approaches.

BACKGROUND: Strategies for development of anti-parasite chemotherapy involve identification of active principles of plants, investigation of drugs already licensed for other pathologies, or validation of specific targets identified within key metabolic pathways. OBJECTIVE: To review the state of the art of drug targets against Trypanosoma cruzi with emphasis on sterol metabolism, kinetoplast DNA (kDNA) sites, trypanothione reductase, cysteine proteinase, hypoxanthine-guanine phosphoribosyltransferase, dihydrofolate reductase and glyceraldehyde-3-phosphate dehydrogenase. METHODS: Current knowledge, accumulated over the last three decades, on targets for design and development of new trypanocidal compounds is described. RESULTS/CONCLUSION: There is an urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox only allopurinol and a few sterol inhibitors have moved to clinical trials, despite the long list of natural and synthetic compounds assayed against T. cruzi. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity associated with a lack of definitive preclinical evidence of parasitological cure in animal models.

Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids.

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.

Mechanism of action of anti-proliferative lysophospholipid analogues against the protozoan parasite Trypanosoma cruzi: potentiation of in vitro activity by the sterol biosynthesis inhibitor ketoconazole.

We investigated the mechanism of action of metabolically stable lysophospholipid analogues (LPAs), with potent anti-tumour and anti-protozoal activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Against the axenically grown epimastigote form of the parasite, the IC(50)s after 120 h for ET-18-OCH(3), miltefosine and ilmofosine were 3, 1 and 3 microM, respectively; at higher concentrations immediate lytic effects were observed. Eradication of the intracellular amastigote, grown inside Vero cells, was achieved at 0.1, 0.1 and 1 microM for ET-18-OCH(3), miltefosine and ilmofosine, respectively. Analysis of the lipid composition of epimastigotes exposed to LPAs at their IC(50) for 120 h showed that the ratio of phosphatidyl-choline (PC) to phosphatidylethanolamine (PE) changed from 1.5 in control cells to c. 0.67 in those treated with the analogues. A significant increase in the content of phosphatidylserine was also observed in treated cells. Intact epimastigotes efficiently incorporated radioactivity from L-[methyl-(14)C]methionine into PC, but not from [methyl-(14)C]choline. ET-18-OCH(3) inhibited the incorporation of L-[methyl-(14)C]methionine into PC with an IC(50) of 2 microM, suggesting that inhibition of the de novo synthesis through the Greenberg's pathway was a primary effect underlying the selective anti-parasitic activity of this compound. Antiproliferative synergism was observed as a consequence of combined treatment of epimastigotes with ET-18-OCH(3) and ketoconazole, a sterol biosynthesis inhibitor, probably due to the fact that a secondary effect of the latter is also a blockade of PC synthesis at the level of PE-PC-N-methyl-transferase.

Chemotherapy of Chagas' disease: the how and the why.

Current developments in experimental chemotherapy of Chagas' disease are reviewed, in particular the demonstration that fourth-generation azole derivatives (inhibitors of sterol C14alpha demethylase), with particular selectivity against Trypanosoma cruzi and special pharmacokinetic properties, are capable of inducing radical parasitological cures in murine models of both acute and chronic disease. These are the first reports of parasitological cure of this disease in its chronic phase. We also discuss the relevance of etiological treatment in the clinical outcome of patients with chronic Chagas' disease. Although previous studies have suggested an important autoimmune component in the pathogenesis of this disease, recent results obtained using highly sensitive polymerase chain reaction based detection methods and detailed immunological characterization of the inflammatory process associated with chagasic cardiomyopathy indicate a positive correlation between tissue parasitism and the severity of cardiac pathological findings. Effective antiparasitic treatment can lead to regression of the inflammatory heart lesions and fibrosis in experimental animals and to stop the progression of the disease in humans. Taken together, these findings support the notion that the presence of the parasite is a necessary and sufficient condition for chagasic cardiomyopathy and confirm the importance of specific etiological treatment in the management of chronic chagasic patients.

Altered lipid composition and enzyme activities of plasma membranes from Trypanosoma (Schizotrypanum) cruzi epimastigotes grown in the presence of sterol biosynthesis inhibitors.

The accepted mechanism for the antiproliferative effects of sterol biosynthesis inhibitors (SBI) against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, is the depletion of specific parasite sterols that are essential growth factors and cannot be replaced by cholesterol, the main sterol present in the vertebrate host. However, the precise metabolic roles of these specific parasite sterols are unknown. We approached this problem by subjecting T. cruzi epimastigotes to two types of SBI, inhibitors of sterol C-14 demethylase and delta 24(25) methyl transferase, and investigating the modification of lipid composition and enzyme activities in the plasma membranes of the parasite. We found in purified plasma membrane from SBI-treated cells that, together with the expected changes in the sterol composition, there was also an inversion of the phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio and a large increase in the content of saturated fatty acids esterified to phospholipids. The modification of the phospholipid headgroup composition correlated with a 70% reduction in the specific activity of the membrane-bound PC-PE-N-methyl transferase SBI-treated cells; it was shown that this inhibition was not due to a direct effect of the drug on the enzyme. Finally, the specific activity of the Mg(2+)-dependent, vanadate-sensitive ATPase present in the membranes was also inhibited by ca. 50% in SBI-treated cells. The results suggest that one of the primary effects of the depletion of endogenous sterols induced by SBI in T. cruzi is a modification of the cellular phospholipid composition as a consequence of a reduced activity of PE-PC-N-methyl transferase and probably of the acyl delta 9 and delta 6 desaturases; this, in turn, could affect the activity of other enzymatic and transport proteins.