In Vitro Trypanocidal and Antibacterial Activities of Essential Oils from Four Species of the Family Annonaceae

The objective of this study was to evaluate the chemical composition, and the trypanocidal and antibacterial activities of the essential oils from four species of Annonaceae: Bocageopsis multiflora (Mart .) R.E.Fr ., Duguetia quitarensis Benth ., Fusaea longifolia (Aubl. ) Saff ., and Guatteria punctata (Aubl .) R.A.Howard . The chemical composition of the essential oils from the aerial parts yielded 23, 20, 21 and 23 constituents, respectively, which were identified by GC/MS. The trypanocidal activity was evaluated against the amastigote and trypomastigote forms of T. cruzi. The antibacterial activity was evaluated by the microdilution method against enterohemorrhagic Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, Streptococcus pyogenes, and methicillin‐resistant Staphylococcus aureus. The results of trypanocidal activity showed that the essential oils of the four species were active at the tested concentrations, with G. punctata essential oil being the most active, with IC₅₀=0.029 μg/mL, and selectivity index (SI)=32, being 34 times more active than the reference drug benznidazole. All EOs showed strong antibacterial activity (minimum inhibitory concentrations of 4.68–37.5 μg/mL) against strains of S. mutans.     

Effect of secochiliolide acid isolated from the Patagonian shrub Nardophyllum bryoides as active component in antifouling paints

Environmental concerns about the use of toxic antifoulants have led to an increased interest in the development of new alternatives. So far, most of the antifouling natural products have been obtained from marine organisms. However, some secondary metabolites from terrestrial plants could be promising antifoulant candidates. The antifouling performance of secochiliolide acid, the main component isolated from Nardophyllum bryoides ethanolic extract, was evaluated for inclusion in rosin-based coatings.Field testing was conducted during the summer months at Mar del Plata harbor, Argentina. The results indicated that secochiliolide acid-based paints completely inhibited the settlement of Bugula neritina colonies, Polydora sp., Hydroides elegans, Corophium sp. and solitary ascidians, and also reduced the attachment of some algae as Enteromorpha intestinalis and Ectocarpus sp. In addition, a lower density and diversity of microfouling species was registered.These results highlighted the importance of terrestrial plants as a sustainable source of potential environmentally friendly antifoulants.     

Hydroxamic acid derivatives: a promising scaffold for rational compound optimization in Chagas disease

This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular parasites and consequently showed activity on the multiplication of the parasite. Treatment of cardiomyocytes and macrophages with the compounds revealed no significant loss in cell viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected by Trypanosoma cruzi with the IC₅₀ value of HAD1 revealed alterations to amastigotes, showing initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of the drug to permeate through the host cell membrane as well as its selectivity to the parasite target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease the activity of metallopeptidases     

Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis

AIMS: The chemical composition of ethanol extracts from a Brazilian (Et-Bra) and a Bulgarian (Et-Blg) propolis, and their activity against the protozoan Trypanosoma cruzi, several fungi and bacteria species were determined. METHODS AND RESULTS: The chemical composition was determined by high temperature high resolution gas chromatography coupled to mass spectrometry. Microbiological activity was assayed in vitro against T. cruzi, Candida albicans, Sporothrix schenckii, Paracoccidioides brasiliensis, Neisseria meningitidis, Streptococcus pneumoniae and Staphylococcus aureus. CONCLUSIONS: Et-Bra and Et-Blg, although with totally distinct compositions, were active against T. cruzi and the three species of fungi. Et-Blg was more effective than Et-Bra against bacteria, particularly N. meningitidis and Strep. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: Although with different classes of components, both propolis extracts showed microbicidal activity. For the bactericidal activity it was possible to establish a positive correlation with the high content of flavonoids of the Bulgarian extract.     

Synthesis and anti-Trypanosoma cruzi activity of derivatives from nor-lapachones and lapachones

New naphthoquinone derivatives were synthesized and assayed against bloodstream trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas’ disease. The compounds were rationalized based on hybrid drugs and appear as important compounds against this parasite. From nor-lapachol were prepared five substituted ortho-naphthofuranquinones, a non-substituted para-naphthofuranquinone, a new oxyrane and an azide and from -lapachone a new non-substituted para-naphthofuranquinone. Other five substituted ortho-naphthofuranquinones recently designed as cytotoxic, were also evaluated. The most active compounds were the ortho naphthofuranquinones 3-(4-methoxyphenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione and 3-(3-nitrophenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione with trypanocidal activity higher than that of benznidazole, the standard drug. The compounds were rationalized based on hybrid drugs and appear as important compounds against T. cruzi. The trypanocidal activity of these substances endowed with redox properties representing a good starting point for a medicinal chemistry program aiming the chemotherapy of Chagas’ disease.     

Changes in fatty acid composition associated with differentiation of Trypanosoma cruzi

The fatty acid composition during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes (metacyclogenesis) was analysed by gas-liquid chromatography and mass spectrometry. Significant qualitative and quantitative changes in the fatty acid composition occurred during incubation of epimastigotes derived from LIT medium in the triatomine artificial urine (TAU). Metacyclogenesis was also followed by alterations in the fatty acid pattern but these were considerably less pronounced when compared to the pattern obtained for TAU-incubated epimastigotes. These results suggest that changes in the lipid composition precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Lactose derivatives are inhibitors of Trypanosoma cruzi trans-sialidase activity toward conventional substrates in vitro and in vivo

Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.     

Ceramide glycosylation and fatty acid hydroxylation influence serological reactivity in Trypanosoma cruzi glycosphingolipids

Ceramide mono (CMH) or dihexoside (CDH) fractions from Trypanosoma cruzi (Dm28c clone) were identified as glucosyl and lactosylceramides containing non-hydroxylated fatty acids. The di-glycosylated form was much more efficiently recognized by sera from T. cruzi-immunized rabbits, indicating that glycosylation influences antigenicity. Fatty acid hydroxylation was also a determinant of serological reactivity, since an alpha-hydroxylated CMH, only present at the Y clone, was recognized by the hyperimmune sera. In summary, these data indicate that T. cruzi CMHs with non-hydroxylated fatty acids are unable to induce antibody responses in animal hosts, which is reverted by the addition of a sugar residue or an alpha-hydroxyl group.     

Synthesis and antitrypanosomal profile of new functionalized 1,3,4-thiadiazole-2-arylhydrazone derivatives, designed as non-mutagenic megazol analogues

In this work we reported the synthesis and the trypanocidal profile of new 1,3,4-thiadiazole-2-arylhydrazone derivatives of nitroimidazole series (4) or phenyl series (5), designed by exploring the molecular hybridization approach between megazol (2) and guanyl hydrazone derivative (3). The evaluation of the activity against bloodstream trypomastigote forms of Trypanosoma cruzi forms lead us to identify a new potent trypamomicide prototype, that is, brazilizone A (4k), which present an IC₅₀/24 h = 5.3 μM.     

Trypanosoma cruzi and human ubiquitin are immunologically distinct proteins despite only three amino acid difference in their primary sequence

The high similarity between Trypanosoma cruzi and human ubiquitin prompted us to characterize the human humoral immunity to host and parasite ubiquitin in Chagas disease and its possible role in Chagas autoimmunity. We have used a simplified one step purification procedure to partially purify T. cruzi ubiquitin. Using this preparation we have performed ELISA and Western blots, to show that chagasic sera recognise T. cruzi but not human or Leishmania ubiquitin indicating a species-specific response. Our results show that despite the high degree of similarity in the primary structure of human and T. cruzi ubiquitins, the three amino acid difference is sufficient to distinguish parasite versus host proteins.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Study of antileishmanial activity of 2-aminobenzoyl amino acid hydrazides and their quinazoline derivatives

A new small library of 2-aminobenzoyl amino acid hydrazide derivatives and quinazolinones derivatives was synthesized and fully characterized by IR, NMR, and elemental analysis. The activity of the prepared compounds on the growth of Leishmania aethiopica promastigotes was evaluated. 2-Benzoyl amino acid hydrazide showed higher inhibitory effect than the quinazoline counterpart. The in vitro antipromastigote activity demonstrated that compounds 2a, 2b, 2f and 4a had IC₅₀ better than standard drug miltefosine and comparable activity to amphotericin B deoxycholate, which indicates their high antileishmanial activity against Leishmania. aethiopica. Among the prepared compounds; 2-amino-N-(6-hydrazinyl-6-oxohexyl)benzamide 2f (IC₅₀ = 0.051 μM) has the best activity, 154 folds more active than reference standard drug miltefosine (IC₅₀ = 7.832 μM), and half fold the activity of amphotericin B (IC₅₀ = 0.035 μM). In addition, this compound was safe and well tolerated by experimental animals orally up to 250 mg/kg and parenterally up to 100 mg/kg.     

Synthesis,antiproliferative and antitrypanosomal activities,and DNA binding of novel 6-amidino-2-arylbenzothiazoles

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC₅₀ = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC₉₀ = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents.     

Anomalous electrophoretic behaviour of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi in relation to its apparent molecular mass

The molecular mass of cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, is 36.3 kDa as calculated from its sequence; this value can increase to about 41 kDa if the three potential N-glycosylation sites are glycosylated in vivo. Yet the apparent molecular mass of the enzyme, as determined by SDS-polyacrylamide gel electrophoresis, has been reported in a range of values from 60 to 40 kDa. We show that the purified enzyme had apparent molecular masses ranging from 51 to 33 kDa, depending on the experimental conditions. This variation is likely to be due to both N-glycosylation and the presence of several disulfide bridges, which make electrophoretic mobility dependent on acrylamide concentration, and reduction and/or boiling of the sample.     

Proteolytic Activities in Cell Extracts of Trypanosoma cruzi*

SYNOPSIS. Cell extracts of culture forms of Trypanosoma cruzi are capable of hydrolysing substances belonging to 4 different groups of protease substrates: (a) substrates for trypsin-like enzymes: benzoyl-arginine-p-nitroanilide and benzoylarginine-naphtylamide: (b) substrates for aminopeptidases: leucyl. lysyl and glutamyl-β-naphtylamide; (c) a substrate for chymotrypsin-like enzymes: carbobenzoxy-L-tyrosine-p-nitrophenylester, and (d) a nonspecific substrate for a broad range of proteases: azocasein. Some physico-chemical characteristics of each enzymic reaction were studied. They were found to be distinct enough to allow attributing each hydrolytic activity to a separate enzyme.     

NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.     

The in vitro trypanocidal activity of N-substituted p-benzoquinone imines: assessment of biochemical structure-activity relationships using the Hansch approach

It has previously been found that naphthoquinones can potentiate the rate of hydrogen peroxide production by mitochondrial preparations of Trypanosoma brucei brucei and that organisms treated with naphthoquinones are more susceptible to lysis, especially in the presence of compounds such as heme, which promote the homolytic cleavage of hydrogen peroxide. We have evaluated the lytic effect of various N-substituted p-benzoquinone imines both in vitro and in vivo and have attempted to correlate their structure with trypanocidal activity using the Hansch approach. While none of the compounds tested proved to be active in vivo, all caused the lysis of trypanosomes in vitro. The parameters that correlated best with trypanocidal activity were the conditional redox potential, the lipophilicity of the substituent attached to the nitrogen atom and the number of active hydrogens on the quinonoid ring. These findings suggest two possible modes of action, which may in fact be related. Conjugate nucleophilic addition and/or oxidative damage could be responsible for lysis of the parasites. These same compounds were previously found to be active against the ascitic sarcoma 180 in mice. The strong correlation between antineoplastic activity in vivo and trypanocidal activity in vitro suggests a similar mode of action in both cases. Further studies aimed at developing a quinonelike compound that will be active against trypanosomes in vivo are now in progress.     

Synthesis and anti-proliferative activity of allogibberic acid derivatives containing 1,2,3-triazole pharmacophore

Sixty novel allogibberic acid derivatives containing 1,2,3-triazole pharmacophore were designed and synthesized. The key chemical processes include aromatization of the A ring in gibberellins, formation of allogibberic azides and its copper mediated Huisgen 1,3-dipolar cycloaddition with alkynes. A number of hybrids containing α,β-unsaturated ketone moiety exhibited excellent in vitro cytotoxic activities. Some of the hybrids were more selective to MCF-7 and SW480 cell lines with IC₅₀ values at least 8-fold more cytotoxic than cisplatin (DDP). The most potent compounds C43 and C45 are more cytotoxic than cisplatin (DDP) against all tested five tumor cell lines, with IC₅₀ values of 0.25–1.72?µM. Mechanism of action studies indicated that allogibberic-triazole derivative C45 could induce the S phase cell cycle arrest and apoptosis in SMMC-7721 cell lines.     

Amino acid composition and N-terminal sequence of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi

Abstract The NADP-linked glutamate dehydrogenase (NADP-GDH) from epimastigotes of Trypanosoma cruzi , Tul 2 stock, has been purified by an improved procedure. The enzyme has subunit molecular weight (47 kDa), amino acid composition and N-terminal sequence similar to those of the NADP-GDH from Escherichia coli , including the N-terminal extension of 15 amino acids present in the E. coli enzyme, but not in the NADP-GDH from Neurospora crassa .