Trypanoside,anti-tuberculosis,leishmanicidal, and cytotoxic activities of tetrahydrobenzothienopyrimidines

The synthesis of 2-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone-derivatives (BTPs) and their in vitro evaluation against Trypanosoma cruzi trypomastigotes, Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, and six human cancer cell lines is described. The in vivo activity of the most active and least toxic compounds against T. cruzi and L. amazonensis was also studied. BTPs constitute a new family of drug leads with potential activity against infectious diseases. Due to their drug-like properties, this series of compounds can potentially serve as templates for future drug-optimization and drug-development efforts for use as therapeutic agents in developing countries.     

Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 microM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16-66 microM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.     

Ethanolic extract of mango (Mangifera indica L.) malformed inflorescence as effective antifungal agent

Mango (Mangifera indica L.) suffers from floral and vegetative malformation and crop production is seriously affected. The anti-fungal activity of ethanolic extract of malformed mango inflorescence was observed at different concentrations (1000, 2000, 3000, 4000 and 5000 μl/ml) against 10 fungi, viz., Ustilago cynodontis, Cercospora cajani, Sphaerotheca sp., Cercospora sp., Alternaria solani, Bipolaris sp., Helminthosporium sp., Curvularia sp., Fusarium udum and Alternaria cajani. Spore germination of most of the fungi was inhibited at 5000 μg/ml. Some of them were also susceptible at 3000 or 4000 μg/ml concentration. Analysis of phenolic acids by high performance liquid chromatography (HPLC) showed 18 peaks in the extract, but only four could be identified, viz., capachin, gallic, benzoic and cinnamic acids. Because of the high efficacy of ethanolic extract of malformed mango inflorescence, its use under field conditions to control some plant diseases has been suggested.     

Fluorine-containing aryloxyethyl thiocyanate derivatives are potent inhibitors of Trypanosoma cruzi and Toxoplasma gondii proliferation

As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas' disease), and Toxoplasma gondii, the etiological agent of toxoplasmosis. This thiocyanate derivative had previously proven to be an effective agent against T. cruzi proliferation. Fluorine-containing thiocyanate derivatives 2 and 3 were threefold more potent than our lead drug 1 against intracellular T. cruzi. The biological evaluation against T. gondii was also very promising. The IC(50) values corresponding to 2 and 3 were at the very low micromolar level against tachyzoites of T. gondii. Both of these drugs are interesting examples of effective antiparasitic agents that have outstanding potential not only as lead drugs but also to be used for further in vivo studies.     

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.     

Jatrorrhizine and columbamine alkaloids isolated from Argemone mexicana are inhibitory to spore germination of some plant pathogenic fungi

The isomeric alkaloids jatrorrhizine and columbamine isolated from the whole plant of Argemone mexicana were assessed against spore germination of some fungi, e.g. Alternaria cajani, Bipolaris sp., Helminthosporium sp., Fusarium udum and Curvularia sp. Both the alkaloids were effective against most of the fungi tested. Jatrorrhizine was highly effective against spore germination of Bipolaris sp., Fusarium udum and Curvularia sp. at 5000 ppm concentration, whereas columbamine was highly effective against Alternaria cajani, Helminthosporium sp. and Fusarium udum at 5000 ppm.     

Synthesis and evaluation of 9,9-dimethylxanthene tricyclics against trypanothione reductase, Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani

Derivatives of 9,9-dimethylxanthene were synthesised and evaluated against trypanothione reductase (TR) and in vitro against parasitic trypanosomes and leishmania. High in vitro antiparasitic activity was observed for some derivatives with one compound showing high activity against all three parasites (ED50 values of 0.02, 0.48 and 0.32 microM, for Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani, respectively). The lack of correlation between inhibitory activity against TR and ED50 values suggests that TR is not the target.     

Antifungal activity of the mixture of quaternary alkaloids isolated from Argemone mexicana against some phytopathogenic fungi

The anti-fungal activity of a mixture of quaternary alkaloids of Argemone mexicana was observed at different concentrations (1000, 2000, 3000, 4000 and 5000 μl/ml) against 10 fungi, viz., Ustilago cynodontis, Cercospora cajani, Sphaerotheca sp., Cercospora sp., Alternaria solani, Bipolaris sp., Helminthosporium sp., Curvularia sp., Fusarium udum and Alternaria cajani. Spore germination was inhibited at 2000, 3000, 4000, 5000 μg/ml. Analysis of phenolics by high performance liquid chromatography (HPLC) recorded 11 peaks in the alkaloids but only three could be identified, viz., tannic, caffeic and ferulic acids. The significant efficacy of the alkaloid under in vitro conditions may open the possibility of its use by farmers under field conditions for controlling some crop diseases.     

Cloning and expression of trypanothione reductase from a New World Leishmania species

Trypanothione disulfide (T[S]₂), an unusual form of glutathione found in parasitic protozoa, plays a crucial role in the regulation of the intracellular thiol redox balance and in the defense against oxidative stress. Trypanothione reductase (TR) is central to the thiol metabolism in all trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania. Here we report the cloning, sequencing and expression of the TR encoding gene from L. (L.) amazonensis. Multiple protein sequence alignment of all known trypanosomatid TRs highlights the high degree of conservation and illustrates the phylogenetic relationships. A 3D homology model for L. amazonensis TR was constructed based on the previously reported Crithidia fasciculata structure. The purified recombinant TR shows enzyme activity and in vivo expression of the native enzyme could be detected in infective promastigotes, both by Western blotting and by immunofluorescence. Nucleotide sequence data reported in this paper is available in the GenBank^TM database under accession number DQ530259.     

Synthesis of 5,5'-dithiobis(2-nitrobenzamides) as alternative substrates for trypanothione reductase and thioredoxin reductase: a microtiter colorimetric assay for inhibitor screening

Trypanothione reductases (TR; EC 1.6.4.8) and thioredoxin reductases (TrxR; EC 1.6.4.5.) are enzymes central to cellular thiol metabolism. Trypanosoma cruzi TR (TcTR) is therefore considered as a potential candidate for drug design against trypanosomiasis. Inhibition of human TrxR (hTrxR) is likely to be beneficial in psoriasis, cancer, and autoimmune diseases, while inhibition of a putative TrxR from Plasmodium falciparum (PfTrxR) might prove effective against malaria. The natural substrates of the first two enzymes are very expensive and difficult to obtain; in the case of PfTrxR, the physiological substrate has not yet been identified. We have therefore synthesized and tested three different 5,5'-dithiobis(2-nitrobenzamides) as alternative substrates of the above enzymes. As with 5, 5'-dithiobis(2-nitrobenzoate) (DTNB), which can be reduced by TRs and TrxRs, the new compounds are converted to their corresponding chromophoric thiolates; however, they have much lower Km values and are therefore less likely to interfere with inhibitor testing. Using the new substrates, a novel enzyme assay has been developed which is identical for all three enzymes, can be performed in a microtiter plate, and is amenable to automation. Thus, the assay provides a versatile and inexpensive tool for kinetic studies and high-throughput inhibitor screening.     

Antifouling activities of octocorals on some marine microfoulers

The antifouling activities of vacuum-dried 70% aqueous alcohol extracts of four gorgonian and five soft corals against four dominant marine fouling diatoms (Navicula subinflata Grun, N. crucicula Smith, Amphora sp., Nitzschia sp.) are described. Of the 36 possible combinations (9 corals x 4 diatoms) 23 of the interactions (64%) showed 100% activity. Extracts of the gorgonian coral Echinogorgia complexa Nutting and the soft coral Dendronephthya (Morchellana) sp. showed 100% growth inhibition against all four fouling diatoms, implying the presence of potent broad spectrum antifouling compounds; other extracts showed more limited species specificity. Exposed cells when transferred to extract-free media, resumed normal growth indicating a nontoxic way of action. The effective concentration for 50% growth inhibition of the attached cells (EC50) varied for each extract and test organism used. The EC50 values for the extract of the gorgonian coral E. complexa against the fouling diatoms ranged from 86 microg/ml (N. subinflata) to 505 microg/ml (Amphora sp.) whereas the EC50 values for extracts of the soft coral Dendronephthya (Morchellana) sp. varied from 28 microg/ml (N. crucicula) to 415 microg/ml (Amphora sp.). The results support the hypothesis that octocorals contain antifouling agents, which could be exploited for the development of nontoxic natural antifouling technology.     

Physcion from marine-derived fungus Microsporum sp. induces apoptosis in human cervical carcinoma HeLa cells

Recently, the relationship between apoptosis and cancer has been emphasized and the induction of apoptosis is recognized as one of the key mechanisms of anti-cancer agents. Marine-derived fungi are valuable sources of structurally diverse bioactive anticancer agents. In the present study, a marine-derived fungus, Microsporum sp. was cultured and an anthraquinone derivative, physcion (11.8 mg) was isolated from the culture broth extract (1710 mg). Physcion has shown cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction in HeLa cells was investigated by the expressions of p53, p21, Bax, Bcl-2, caspase-9, and caspase-3 proteins. The Western blot analysis has revealed that physcion could significantly induce cell apoptosis through down-regulating of Bcl-2 expression, up-regulating of Bax expression, and activating the caspase-3 pathway. Furthermore, physcion induced the formation of reactive oxygen species (ROS) in HeLa cells. Collectively, these results suggest that physcion could be a potential candidate in the field of anticancer drug discovery against human cervical cancer.     

Leishmanicidal Metabolites from Cochliobolus sp., an Endophytic Fungus Isolated from Piptadenia adiantoides (Fabaceae)

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman''s reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL⁻¹. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC₅₀ values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.     

Design and preparation of sterol mimetics as potential antiparasitics

We have previously shown that azasterols have activity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania species, which are the causative agents of various neglected tropical diseases. In this paper, we discuss the replacement of the sterol core of the azasterols with sterol mimics. Various mimics were designed, and the structures were minimised to see if they could adopt a similar conformation to that of the azasterols. From this, two series of mimics were synthesised and then evaluated against the parasites. Compounds showed moderate activity.     

High-throughput screening affords novel and selective trypanothione reductase inhibitors with anti-trypanosomal activity

Trypanothione reductase (TR), an enzyme that buffers oxidative stress in trypanosomatid parasites, was screened against commercial libraries containing approximately 134,500 compounds. After secondary screening, four chemotypes were identified as screening positives with selectivity for TR over human glutathione reductase. Thirteen compounds from these four chemotypes were purchased, and their in vitro activity against TR and Trypanosoma brucei is described.     

Antioxidant and antitopoisomerase activities in plant extracts of some Colombian flora from La Marcada Natural Regional Park

Many plants have been used to treat some diseases and infections since time immemorial, and this potential has been exploited by the pharmaceutical industry in the search of new analgesic, anticarcinogenic and antimicrobial agents, among other active agents. In order to contribute with bioprospection studies on the Colombian flora, 35 extracts from 13 plant species belonging to seven families (Apocynaceae, Cactaceae, Costaceae, Eremolepidaceae, Passifloraceae, Solanaceae and Urticaceae) were collected from La Marcada Natural Regional Park (LMNRP), Colombia. Dichloromethane, n-hexane and aqueous-methanol crude extracts were prepared and evaluated for their activity against Saccharomyces cerevisiae RS322N, R52Y and RS321 strains in the yeast mutant assay and their antioxidant capacity through the DPPH test. The dichloromethane extract from Myriocarpa stipitata (Urticaceae) showed moderate inhibitory activity against the three S. cerevisiae strains tested. The capacity of the dichloromethane extract from M. stipitata to inhibit the enzyme topoisomerase I and to cause DNA damage was inferred from these results. In the DPPH assay, the n-hexane crude extract from Costus sp. (Costaceae) showed good antioxidant activity (48%); in addition, the crude dichloromethane and aqueous-methanol extracts from Rhipsalis micrantha (Cactaceae) showed moderate antioxidant activity with percentage of 29 and 21%, respectively.     

Antiparasitic activity and effect of casearins isolated from Casearia sylvestris on Leishmania and Trypanosoma cruzi plasma membrane

Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi, respectively, and are included among the most neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G, and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with IC₅₀ values ranging from 0.53 to 2.77 μg/ml. Leishmania infantum promastigotes were also susceptible to casearins, with IC₅₀ values in a range between 4.45 and 9.48 μg/ml. These substances were also evaluated for mammalian cytotoxicity against NCTC cells resulting in 50% cytotoxic concentrations (CC₅₀) ranging from 1.46 to 13.76 μg/ml. Additionally, the action of casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug design studies.     

Putative mycotoxin-producing fungi isolated from alpataco (Prosopis flexuosa) fruits

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.     

Endophytic fungi in rose (Rosa hybrida) in Bogota, Colombia

We have investigated the presence of endophytic fungi associated with rose plants (Rosa hybrida) in Colombia. Endophytic fungi were isolated from healthy leaves of ten ornamental roses plants from gardens cultured in malt extract, peptone, yeast extract agar plates (MPY). We sampled 560 leaves fragments, 56 per sample. Endophytic fungi comprised 92 isolates (16.4%); of these isolates, 41 were classified as sterile mycelium (without reproductive structures that allowed their identification), 31 isolates were identified to genus or to species, and 20 isolates could not be identified at all. The identified endophytic fungi were as follow: Nigrospora oryzae, Aureobasidium spp, Acremonium spp. The fungi Nodulisporium sp, Gliocladium virens, Cladosporium sp, Alternaria sp, Phoma sp and Chaetomium globosum were represented by one isolate each. Since the endophytic fungi are known for their capacity to produce metabolites with biological activity, it is possible that the microorganisms found in this study have potential as antagonist of rose pathogens.     

Development of selective media for the isolation and enumeration of Alternaria species from soil and plant debris

A new semi-selective medium, acidified weak potato-dextrose agar (AWPDA) with Mertect (active ingredient: thiabendazole), was developed for the isolation and enumeration of Alternaria species from samples of soil and plant debris. The medium was selected based on growth inhibition tests against Alternaria and several other commonly encountered saprobic fungi utilizing three antifungal agents, Botran (active ingredient: dichloran), Bayleton (active ingredient: triadimefon), and Mertect, and two basal media, acidified potato-dextrose agar (APDA) and AWPDA. Botran inhibited growth of Rhizopus stolonifer moderately, but had little effect on Cladosporium cladosporoides, Fusarium oxysporum, Penicillium chrysogenum, or Trichoderma harzianum. Bayleton inhibited growth of R. stolonifer and C. cladosporoides severely, and inhibited growth of F. oxysporum, P. chrysogenum, and T. harzianum moderately. Mertect inhibited growth of C. cladosporoides, F. oxysporum, P. chrysogenum, and T. harzianum completely, but had little or moderate effect on R. stolonifer. All three antifungal agents inhibited growth of Alternaria species slightly or moderately. The combination of Bayleton and Mertect inhibited growth of all fungi severely. A comparison of recovery rates of Alternaria from soil and plant debris samples on AWPDA with Mertect and weak potato-dextrose agar (WPDA) revealed that Alternaria spp. accounted for 63.6%-81.0% of recovered fungal isolates on AWPDA with Mertect as compared to 0.6%-2.7% of recovered isolates on WPDA. The AWPDA medium with Mertect exhibited superior selective growth of Alternaria species from samples of soil and plant debris, and will be useful in studies where the recovery and enumeration of Alternaria species is necessary.