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Abstract: Human alpha2-macroglobulin (α2M) is a 720kDa glycoprotein that presents two ultrastructural conformations: slow (S-α2M) and fast (F-α2M). α2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of α2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-α2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-α2M. TEM proved to be an important tool to analyze the effect of biochemical changes on α2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the α2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B4 was slightly lower in F-α2M than in S-α2M. Among the neuraminidases used to desialylate both conformations of α2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of α2M molecules, mediated by lectin binding and clearly visualized by TEM. [Copyright &y& Elsevier]

Metronidazole (Mtz) is a commercial broad-spectrum nitroimidazolic derivative with relevant antimicrobial activity and relative safety profile. Therefore, it is fair to consider Mtz a candidate for drug repurposing for other neglected conditions such as Chagas disease (CD), a parasitic pathology caused by Trypanosoma cruzi . CD is treated only with benznidazole (Bz) and nifurtimox, both introduced in clinics decades ago despite important limitations, including low efficacy on the later disease stage (chronic form) and severe side effects. New cheap and fast alternative treatments for CD are needed, thus the repurposing of Mtz was assessed in vitro and in vivo in mono- and combined therapy. In vitro assays demonstrated EC 50 > 200 µM for Mtz, while for Bz the values ranged from 2.51 µM (intracellular forms) to 11.5 µM (bloodstream trypomastigotes). When both drugs were combined in fixed-ratio proportions, Mtz promoted Bz potency (lower EC 50 values). In vivo toxicity assays for Mtz in mice showed no adverse effects neither histopathological alterations up to 2000 mg/kg. Regarding experimental T. cruzi infection, Bz 100 mg/kg suppressed parasitemia while Mtz (up to 1000 mg/kg) in monotherapy did not, but prolonged animal survival at 250 and 500 regimen doses. The combination of both drugs (Bz 10 + Mtz 250) prevented mortality (70%) besides protected against electric cardiac alterations triggered by the parasite infection. Although not able to reduce parasite load, the combination therapy prevented animal mortality; this was possibly due to a protection of the electric cardiac physiology that is normally altered in experimental infection of T. cruzi. It also suggested that the interaction with Mtz could have improved the pharmacokinetics of Bz. Our study emphasizes the importance of drug repurposing and combined therapy for CD to contribute to alternative therapies for this neglected and silent pathology. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12–14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD50 values, good selectivity index, and good activity at 4°C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection. [ABSTRACT FROM AUTHOR]

Abstract: A2M is a broad spectrum proteinase inhibitor and cytokine carrier, besides presenting anti-apoptotic activity through the binding to its receptor, LRP. During Trypanosoma cruzi infection, apoptosis of host cells and intracellular parasites is commonly observed both in vivo and in vitro. Since plasma as well as tissue A2M levels are increased in both murine and human acute T. cruzi infection, we evaluated the possible role of A2M (its methylamine transformed Fast form—A2M-F) in regulating apoptotic events in peritoneal macrophages and cardiomyocytes during in vitro interaction with the parasite. Our data showed that DNA fragmentation (a hallmark of apoptosis) of both host cells and parasites was inhibited by A2M-F. Impaired apoptosis was also noted when A2M-F was added to the cultures maintained under serum deprivation. In addition, macrophages from C57/BL6 mice, known to display higher LRP levels as compared to those of C3H lineage, displayed higher reduction in the apoptotic levels during the A2M-F treatment. [Copyright &y& Elsevier]

Furamidine (DB75) and related unfused aromatic diamidines have proven useful for the treatment of parasitic infections. These compounds were primarily developed to combat infections by Pneumocystis carinii and African trypanosomes but they are also active against other parasites. Here we have investigated the in vitro effects of DB75 and its phenyl-substituted analog DB569 on two kinetoplastid haemoflagellates Trypanosomatidae: Trypanosoma cruzi and Leishmania (L) amazonensis. The phenyl-amidine compound DB569 has equivalent DNA binding properties compared to DB75 but it was selected on the basis of its distinct tumor cell distribution properties. We found that DB569 is significantly more potent than DB75 at reducing the proliferation of the parasites, using either isolated parasites in cultures or with cardiomyocyte and macrophage host cells. DB569 is effective towards the intracellular forms of T. cruzi (IC50 in the low-micromolar range) and it exhibits trypanocidal dose-dependent effects against trypomastigote forms of T. cruzi parasites obtained from the Y strain and Dm28c clone, which belong to two different biodemes. Fluorescence microscopy experiments indicated that both diamidines were mostly localized in the nucleus of the mammalian host cells and within the nuclei and kinetoplast of the parasites. Electron microscopy studies showed that the treatment of the parasites with DB75 and DB569 induces important alterations of the parasite nucleus and kinetoplast, at sites where their DNA target is localized. Altogether, the data suggest that the phenyl-substituted furamidine analogue DB569 is a potential new candidate for the treatment of the Chagas’ disease and Leishmaniasis. [Copyright &y& Elsevier]