RESUMEN
Chagas disease is a neglected tropical parasitic disease caused by the protozoan Trypanosoma cruzi. Worldwide, an estimated 8 million people are infected with T. cruzi, causing more than 10,000 deaths per year. Currently, only two drugs, nifurtimox and benznidazole (BNZ), are approved for its treatment. However, both are ineffective during the chronic phase, show toxicity, and produce serious side effects. This work aimed to obtain and evaluate novel 2-nitroimidazole-N-acylhydrazone derivatives analogous to BNZ. The design of these compounds used the two important pharmacophoric subunits of the BNZ prototype, the 2-nitroimidazole nucleus and the benzene ring, and the bioisosterism among the amide group of BNZ and N-acylhydrazone. The 27 compounds were obtained by a three-step route in 57%-98% yields. The biological results demonstrated the potential of this new class of compounds, since eight compounds were potent and selective in the in vitro assay against T. cruzi amastigotes and trypomastigotes using a drug-susceptible strain of T. cruzi (Tulahuen) (IC50 = 4.3-6.25 µM) and proved to be highly selective with low cytotoxicity on L929 cells. The type I nitroreductase (TcNTR) assay suggests that the new compounds may act as substrates for this enzyme.
Asunto(s)
Hidrazonas , Nitroimidazoles , Pruebas de Sensibilidad Parasitaria , Tripanocidas , Trypanosoma cruzi , Trypanosoma cruzi/efectos de los fármacos , Tripanocidas/farmacología , Tripanocidas/síntesis química , Tripanocidas/química , Nitroimidazoles/farmacología , Nitroimidazoles/química , Nitroimidazoles/síntesis química , Relación Estructura-Actividad , Animales , Hidrazonas/farmacología , Hidrazonas/síntesis química , Hidrazonas/química , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Ratones , Estructura Molecular , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.
Asunto(s)
Enfermedad de Chagas , Nitroimidazoles , Tripanocidas , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/metabolismo , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular , Mutágenos/farmacología , Tripanocidas/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Triazoles/química , Nitrorreductasas/metabolismoRESUMEN
BACKGROUND: Chagas disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Current therapeutic management is limited to treatment with nitroheterocyclic drugs, such as nifurtimox (NFX) and benznidazole (BZ). Thus, the identification of affordable and readily available drugs to treat resistant parasites is urgently required worldwide. To analyse the effects of BZ on human macrophage gene expression, a quantitative PCR (qPCR) array analysis was performed using drug transporter and oxidative stress pathway genes to compare the gene expression profiles of human differentiated THP-1 macrophage (THP-1 MΦ) cells infected or not with benznidazole-sensitive (CL Brener) and naturally benznidazole-resistant (Colombiana) T. cruzi parasites followed by treatment with BZ. RESULTS: The gene expression analysis indicated that the expression levels of 62 genes were either up- or downregulated at least 3-fold in the host upon infection with CL Brener and BZ treatment, of which 46 were upregulated and 16 were downregulated. Moreover, the expression level of 32 genes was altered in THP-1 MФ cells infected with Colombiana and treated with BZ, of which 29 were upregulated and 3 were downregulated. Our results revealed that depending on the specific condition, human THP-1 MΦ cells infected with T. cruzi strains with sensitive or resistant phenotypes and treated with BZ expressed high mRNA levels of AQP1, AQP9 and ABCB1 (MDR1) compared to those of the control cells. CONCLUSIONS: Our findings suggest that the proteins encoded by AQP1, AQP9 and ABCB1 may be implicated in benznidazole detoxification. Therefore, studies on gene expression are required to better understand the host response to pathogens and drug treatment integrated with functional and metabolic data to identify potentially novel targets for the treatment of this important and neglected tropical disease.
Asunto(s)
Resistencia a Medicamentos , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Nitroimidazoles/farmacología , Estrés Oxidativo , Trypanosoma cruzi/efectos de los fármacos , ADN Protozoario/genética , Expresión Génica , Humanos , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Células THP-1RESUMEN
BACKGROUND: Chagas disease, also known as American trypanosomiasis, is classified as one of the 17 most important neglected diseases by the World Health Organization. The only drugs with proven efficacy against Chagas disease are benznidazole and nifurtimox, however both show adverse effects, poor clinical efficacy, and development of resistance. For these reasons, the search for new effective chemical entities is a challenge to research groups and the pharmaceutical industry. OBJECTIVE: Synthesis and evaluation of antitrypanosomal activities of a series of thiosemicarbazones and semicarbazones containing 1,2,3-1H triazole isatin scaffold. METHOD: 5'-(4-alkyl/aryl)-1H-1,2,3-triazole-isatins were prepared by Huisgen 1,3-dipolar cycloaddition and the thiosemicarbazones and semicarbazones were obtained by the 1:1 reactions of the carbonylated derivatives with thiosemicarbazide and semicarbazide hydrochloride, respectively, in methanol, using conventional reflux or microwave heating. The compounds were assayed for in vitro trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Beyond the thio/semicarbazone derivatives, isatin and triazole synthetic intermediates were also evaluated for comparison. RESULTS: A series of compounds were prepared in good yields. Among the 37 compounds evaluated, 18 were found to be active, in particular thiosemicarbazones containing a non-polar saturated alkyl chain (IC50 = 24.1, 38.6, and 83.2 µM; SI = 11.6, 11.8, and 14.0, respectively). To further elucidate the mechanism of action of these new compounds, the redox behaviour of some active and inactive derivatives was studied by cyclic voltammetry. Molecular docking studies were also performed in two validated protein targets of Trypanosoma cruzi, i.e., cruzipain (CRZ) and phosphodiesterase C (TcrPDEC). CONCLUSION: A class of thio/semicarbazones structurally simple and easily accessible was synthesized. Compounds containing thiosemicarbazone moieties showed the best results in the series, being more active than the corresponding semicarbazones. Our results indicated that the activity of these compounds does not originate from an oxidation-reduction pathway but probably from the interactions with trypanosomal enzymes.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Técnicas Electroquímicas/métodos , Semicarbazonas/síntesis química , Semicarbazonas/farmacología , Análisis Espectral/métodos , Tiosemicarbazonas/síntesis química , Tiosemicarbazonas/farmacología , Tripanocidas/síntesis química , Tripanocidas/farmacología , Animales , Línea Celular , Ratones , Simulación del Acoplamiento Molecular , Semicarbazonas/química , Relación Estructura-Actividad , Tiosemicarbazonas/química , Tripanocidas/química , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Eukaryotic initiation factor 5A (eIF5A) is a conserved protein with an essential role in translation elongation. Using one and two-dimensional western blotting, we showed that the eIF5A protein level was 2-fold lower in benznidazole (BZ)-resistant (BZR and 17LER) Trypanosoma cruzi populations than in their respective susceptible counterparts (BZS and 17WTS). To confirm the role of eIF5A in BZ resistance, we transfected BZS and 17WTS with the wild-type eIF5A or mutant eIF5A-S2A (in which serine 2 was replaced by alanine). Upon overexpressing eIF5A, both susceptible lines became approximately 3- and 5-fold more sensitive to BZ. In contrast, the eIF5A-S2A mutant did not alter its susceptibility to BZ. These data suggest that BZ resistance might arise from either decreasing the translation of proteins that require eIF5A, or as a consequence of differential levels of precursors for the hypusination reactions (e.g., spermidine and trypanothione), both of which alter BZ's effects in the parasite.
Asunto(s)
Resistencia a Medicamentos/genética , Nitroimidazoles/farmacología , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Expresión Génica , Humanos , Factores de Iniciación de Péptidos/análisis , Factores de Iniciación de Péptidos/efectos de los fármacos , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/efectos de los fármacos , Trypanosoma cruzi/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Eukaryotic initiation factor 5A (eIF5A) is a conserved protein with an essential role in translation elongation. Using one and two-dimensional western blotting, we showed that the eIF5A protein level was 2-fold lower in benznidazole (BZ)-resistant (BZR and 17LER) Trypanosoma cruzi populations than in their respective susceptible counterparts (BZS and 17WTS). To confirm the role of eIF5A in BZ resistance, we transfected BZS and 17WTS with the wild-type eIF5A or mutant eIF5A-S2A (in which serine 2 was replaced by alanine). Upon overexpressing eIF5A, both susceptible lines became approximately 3- and 5-fold more sensitive to BZ. In contrast, the eIF5A-S2A mutant did not alter its susceptibility to BZ. These data suggest that BZ resistance might arise from either decreasing the translation of proteins that require eIF5A, or as a consequence of differential levels of precursors for the hypusination reactions (e.g., spermidine and trypanothione), both of which alter BZ's effects in the parasite.
Asunto(s)
Humanos , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Resistencia a Medicamentos/genética , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Nitroimidazoles/farmacología , Trypanosoma cruzi/genética , Expresión Génica , Factores de Iniciación de Péptidos/análisis , Factores de Iniciación de Péptidos/efectos de los fármacos , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/efectos de los fármacosRESUMEN
Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime.
Asunto(s)
Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/patogenicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Meglumina/farmacología , Compuestos Organometálicos/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antioxidantes/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Glutatión Reductasa/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Glutatión Sintasa/metabolismo , Interacciones Huésped-Patógeno , Humanos , Antimoniato de Meglumina , Reacción en Cadena de la PolimerasaRESUMEN
Analogues of 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]indol-4-amine 1, a known cruzain inhibitor, were synthesized using a molecular simplification strategy. Five series of analogues were obtained: indole, pyrimidine, quinoline, aniline and pyrrole derivatives. The activity of the compounds was evaluated against the enzymes cruzain and rhodesain as well as against Trypanosoma cruzi amastigote and trypomastigote forms. The 4-aminoquinoline derivatives showed promising activity against both enzymes, with IC50 values ranging from 15 to 125µM. These derivatives were selective inhibitors for the parasitic proteases, being unable to inhibit mammalian cathepsins B and S. The most active compound against cruzain (compound 5a; IC50=15µM) is considerably more synthetically accessible than 1, while retaining its ligand efficiency. As observed for the original lead, compound 5a was shown to be a competitive enzyme inhibitor. In addition, it was also active against T. cruzi (IC50=67.7µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50=3.1µM) with remarkable selectivity index (SI=128) compared to uninfected fibroblasts. Both 5a and 4b exhibit drug-like physicochemical properties and are predicted to have a favorable ADME profile, therefore having great potential as candidates for lead optimization in the search for new drugs to treat Chagas disease.
Asunto(s)
Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/química , Evaluación Preclínica de Medicamentos , Análisis Espectral/métodos , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimologíaRESUMEN
A series of novel xylitan derivatives derived from xylitol were synthesized using operationally simple procedures. A xylitan acetonide was the key intermediate used to prepare benzoate, arylsulfonate esters and 1,2,3-triazole derivatives of xylitan. These compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity against trypomastigote and amastigote forms of the parasite in T. cruzi-infected cell lineages. Benznidazole was used as positive control against T. cruzi and cytotoxicity was determined in mammalian L929 cells. The arylsulfonate xylitan derivative bearing a nitro group displayed the best activity of all the compounds tested, and was slightly more potent than the reference drug benznidazole. The importance of the isopropylidene ketal moiety was established and the greater lipophilicity of these compounds suggests enhancement in cell penetration.
Asunto(s)
Tripanocidas/síntesis química , Tripanocidas/farmacología , Xilitol/síntesis química , Xilitol/farmacología , Humanos , Pruebas de Sensibilidad Parasitaria , Trypanosoma cruzi/efectos de los fármacos , Xilitol/análogos & derivadosRESUMEN
OBJECTIVE: To evaluate the in vitro anti-Trypanosoma cruzi (T. cruzi) activity of organic extracts prepared from halophyte species collected in the southern coast of Portugal (Algarve), and chemically characterize the most active samples. METHODS: Acetone, dichloromethane and methanol extracts were prepared from 31 halophyte species and tested in vitro against trypomastigotes and intracellular amastigotes of the Tulahuen strain of T. cruzi. The most active extract was fractionated by preparative HPLC-DAD, affording 11 fractions. The most selective fraction was fully characterized by (1)H NMR. RESULTS: From 94 samples tested, one was active, namely the root dichloromethane extract of Juncus acutus (IC50 < 20 µg/mL). This extract was fractionated by HPLC, affording 11 fractions, one of them containing only a pure compound (juncunol), and tested for anti-parasitic activity. Fraction 8 (IC50 = 4.1 µg/mL) was the most active, and was further characterized by (1)H NMR. The major compounds were phenanthrenes, 9,10-dihydrophenanthrenes and benzocoumarins. CONCLUSION: Our results suggest that the compounds identified in fraction 8 are likely responsible for the observed anti parasitic activity. Further research is in progress aiming to isolate and identify the specific active molecules. To the best of our knowledge, this is the first report on the in vitro anti T. cruzi activity of halophyte species.
RESUMEN
Lipoamide dehydrogenase (LipDH) is a flavin-containing disulfide oxidoreductase from the same group of thioredoxin reductase, glutathione reductase and trypanothione reductase. This enzyme is found in the mitochondria of all aerobic organisms where it takes part in at least three important multienzyme complexes from the citric acid cycle. In this study, we performed a phylogenetic analysis comparing the amino acid sequence of the LipDH from Trypanosoma cruzi (TcLipDH) with the LipDH from other organisms. Subsequently, the copy number of the TcLipDH gene, the mRNA and protein levels, and the enzymatic activity of the LipDH were determined in populations and strains of T. cruzi that were either resistant or susceptible to benznidazole (BZ). In silico analysis showed the presence of two TcLipDH alleles in the T. cruzi genome. It also showed that TcLipDH protein has less than 55% of identity in comparison to the human LipDH, but the active site is conserved in both of them. Southern blot results suggest that the TcLipDH is a single copy gene in the genome of the T. cruzi samples analyzed. Northern blot assays showed one transcript of 2.4 kb in all T. cruzi populations. Northern blot and Real Time RT-PCR data revealed that the TcLipDH mRNA levels were 2-fold more expressed in the BZ-resistant T. cruzi population (17LER) than in its susceptible pair (17WTS). Western blot results revealed that the TcLipDH protein level is 2-fold higher in 17LER sample in comparison to 17WTS sample. In addition, LipDH activity was higher in the 17LER population than in the 17WTS. Sequencing analysis revealed that the amino acid sequences of the TcLipDH from 17WTS and 17LER populations are identical. Our findings show that one of the mechanisms associated with in vitro-induced BZ resistance to T. cruzi correlates with upregulation of LipDH enzyme.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/genética , Resistencia a Medicamentos , Nitroimidazoles/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Alelos , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Clonación Molecular , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Dihidrolipoamida Deshidrogenasa/química , Resistencia a Medicamentos/genética , Dosificación de Gen , Regulación Enzimológica de la Expresión Génica , Ratones , Mitocondrias/enzimología , Filogenia , ARN Mensajero/metabolismo , ARN Protozoario/química , ARN Protozoario/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADN , Trypanosoma cruzi/genéticaRESUMEN
Endophytic fungi represent ubiquitous microbial organisms able to live in the tissues of different plants around the world and represent a prolific source of bioactive metabolites. In the present study, the endophytic fungus Aspergillus calidoustus was isolated from the medicinal plant Acanthospermum australe (Asteraceae), and identified using molecular, physiological and morphological methods. A methylene chloride crude extract of A. calidoustus has been produced and subjected to antifungal bioassay-directed fractionation which resulted in the isolation of the two bioactive compounds: ophiobolin K and 6-epi-ophiobolin K. These pure compounds displayed antifungal activity against fungal plant pathogens, protozoal activity against Trypanosoma cruzi, and cytotoxic activity against human tumoral cell lines. The results show that A. calidoustus was able to produce the antifungal and cytotoxic metabolites ophiobolin K and 6-epi-ophiobolin K, which may help the fungus to colonise and occupy the substratum as well as survive in natural environments.
Asunto(s)
Antifúngicos/química , Antimaláricos/química , Antineoplásicos/química , Aspergillus/química , Sesterterpenos/química , Antifúngicos/aislamiento & purificación , Antimaláricos/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Asteraceae/microbiología , Línea Celular Tumoral , HumanosRESUMEN
This study assessed the diversity of cultivable rock-associated fungi from Atacama Desert. A total of 81 fungal isolates obtained were identified as 29 Ascomycota taxa by sequencing different regions of DNA. Cladosporium halotolerans, Penicillium chrysogenum and Penicillium cf. citrinum were the most frequent species, which occur at least in four different altitudes. The diversity and similarity indices ranged in the fungal communities across the latitudinal gradient. The Fisher-α index displayed the higher values for the fungal communities obtained from the siltstone and fine matrix of pyroclastic rocks with finer grain size, which are more degraded. A total of 23 fungal extracts displayed activity against the different targets screened. The extract of P. chrysogenum afforded the compounds α-linolenic acid and ergosterol endoperoxide, which were active against Cryptococcus neoformans and methicillin-resistance Staphylococcus aureus respectively. Our study represents the first report of a new habitat of fungi associated with rocks of the Atacama Desert and indicated the presence of interesting fungal community, including species related with saprobes, parasite/pathogen and mycotoxigenic taxa. The geological characteristics of the rocks, associated with the presence of rich resident/resilient fungal communities suggests that the rocks may provide a favourable microenvironment fungal colonization, survival and dispersal in extreme conditions.
Asunto(s)
Ascomicetos/metabolismo , Cladosporium/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Sedimentos Geológicos/microbiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Penicillium/metabolismo , Ascomicetos/clasificación , Ascomicetos/genética , Ascomicetos/aislamiento & purificación , Chile , Cladosporium/clasificación , Cladosporium/genética , Cladosporium/aislamiento & purificación , Clima Desértico , Ecología , Ecosistema , Datos de Secuencia Molecular , Penicillium/clasificación , Penicillium/genética , Penicillium/aislamiento & purificaciónRESUMEN
The discovery of new antiparasitic compounds against Trypanosoma cruzi, the etiological agent of Chagas disease, is necessary. Novel aryloxy/aryl thiosemicarbazone-based conformationally constrained analogs of thiosemicarbazones (1) and (2) were developed as potential inhibitors of the T. cruzi protease cruzain, using a rigidification strategy of the iminic bond of (1) and (2). A structure-activity relationship analysis was performed in substituents attached in both aryl and aryloxy rings. This study indicated that apolar substituents or halogen atom substitution at the aryl position improved cruzain inhibition and antiparasitic activity in comparison to unsubstituted thiosemicarbazone. Two of these compounds displayed potent inhibitory antiparasitic activity by inhibiting cruzain and consequently were able to reduce the parasite burden in infected cells and cause parasite cell death through necrosis. In conclusion, we demonstrated that conformational restriction is a valuable strategy in the development of antiparasitic thiosemicarbazones.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tiosemicarbazonas/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Tiosemicarbazonas/síntesis química , Tiosemicarbazonas/química , Tripanocidas/síntesis química , Trypanosoma cruzi/enzimologíaRESUMEN
Cysteine metabolism is considered essential for the crucial maintenance of a reducing environment in trypanosomatids due to its importance as a precursor of trypanothione biosynthesis. Expression, activity, functional rescue, and overexpression of cysteine synthase (CS) and cystathionine ß-synthase (CßS) were evaluated in Leishmania braziliensis promastigotes and intracellular amastigotes under in vitro stress conditions induced by hydrogen peroxide (H2O2), S-nitroso-N-acetylpenicillamine, or antimonial compounds. Our results demonstrate a stage-specific increase in the levels of protein expression and activity of L. braziliensis CS (LbrCS) and L. braziliensis CßS (LbrCßS), resulting in an increment of total thiol levels in response to both oxidative and nitrosative stress. The rescue of the CS activity in Trypanosoma rangeli, a trypanosome that does not perform cysteine biosynthesis de novo, resulted in increased rates of survival of epimastigotes expressing the LbrCS under stress conditions compared to those of wild-type parasites. We also found that the ability of L. braziliensis promastigotes and amastigotes overexpressing LbrCS and LbrCßS to resist oxidative stress was significantly enhanced compared to that of nontransfected cells, resulting in a phenotype far more resistant to treatment with the pentavalent form of Sb in vitro. In conclusion, the upregulation of protein expression and increment of the levels of LbrCS and LbrCßS activity alter parasite resistance to antimonials and may influence the efficacy of antimony treatment of New World leishmaniasis.
Asunto(s)
Cistationina betasintasa/genética , Cisteína Sintasa/genética , Leishmania braziliensis/genética , Estrés Oxidativo/fisiología , Proteínas Protozoarias/genética , Regulación hacia Arriba/genética , Antimonio/farmacología , Antiprotozoarios/farmacología , Línea Celular , Humanos , Peróxido de Hidrógeno/farmacología , Leishmania braziliensis/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Estrés Oxidativo/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Trypanosoma rangeli/efectos de los fármacos , Trypanosoma rangeli/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We describe herein the antitrypanosomal activity of 20 novel 1,3-bis(aryloxy)propan-2-amine derivatives. Compounds 2, 4, 6, 12, 15, 16 and 19 were significantly active against amastigote and trypomastigote forms, with half maximal inhibitory concentrationvalues in the range of 6-18 µM. In the cytotoxicity tests against L929 cells, only compound 4 presented selectivity index value above 10, indicating low toxicity.
Asunto(s)
Derivados del Benceno/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Línea Celular , Concentración 50 Inhibidora , Pruebas de Sensibilidad ParasitariaRESUMEN
Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 µg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 µg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 µg/mL), Candida albicans and Candida tropicalis (MIC 64-128 µg/mL). Fourteen extracts at a concentration of 20 µg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 µg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 µg/mL (2.43 µM) in a T. cruzi cellular culture assay.
Asunto(s)
Caesalpinia/microbiología , Depsipéptidos/farmacología , Endófitos/aislamiento & purificación , Fusarium/aislamiento & purificación , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Candida albicans/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Fraccionamiento Químico , Mezclas Complejas , Cartilla de ADN , Depsipéptidos/aislamiento & purificación , Endófitos/clasificación , Enterobacteriaceae/efectos de los fármacos , Fusarium/metabolismo , Bacilos Grampositivos Formadores de Endosporas/efectos de los fármacos , Humanos , Leishmania/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Tripanocidas/aislamiento & purificaciónRESUMEN
We surveyed the diversity and capability of producing bioactive compounds from a cultivable fungal community isolated from oligotrophic soil of continental Antarctica. A total of 115 fungal isolates were obtained and identified in 11 taxa of Aspergillus, Debaryomyces, Cladosporium, Pseudogymnoascus, Penicillium and Hypocreales. The fungal community showed low diversity and richness, and high dominance indices. The extracts of Aspergillus sydowii, Penicillium allii-sativi, Penicillium brevicompactum, Penicillium chrysogenum and Penicillium rubens possess antiviral, antibacterial, antifungal, antitumoral, herbicidal and antiprotozoal activities. Bioactive extracts were examined using (1)H NMR spectroscopy and detected the presence of secondary metabolites with chemical shifts. Our results show that the fungi present in cold-oligotrophic soil from Antarctica included few dominant species, which may have important implications for understanding eukaryotic survival in cold-arid oligotrophic soils. We hypothesize that detailed further investigations may provide a greater understanding of the evolution of Antarctic fungi and their relationships with other organisms described in that region. Additionally, different wild pristine bioactive fungal isolates found in continental Antarctic soil may represent a unique source to discover prototype molecules for use in drug and biopesticide discovery studies.
Asunto(s)
Bioprospección , Frío Extremo , Hongos/aislamiento & purificación , Microbiota , Microbiología del Suelo , Aedes/efectos de los fármacos , Animales , Regiones Antárticas , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/toxicidad , Productos Biológicos/aislamiento & purificación , Productos Biológicos/toxicidad , Citotoxinas/aislamiento & purificación , Citotoxinas/toxicidad , Hongos/química , Hongos/clasificación , Humanos , Insecticidas/aislamiento & purificación , Insecticidas/toxicidad , Lactuca/efectos de los fármacos , Células MCF-7RESUMEN
The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC50s) of ≤ 10 µM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC50 = 1.2 ± 1.0 µM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series.
Asunto(s)
Catepsina B/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Proteínas Protozoarias/metabolismo , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , Tripanocidas/síntesis química , Animales , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Tripanocidas/química , Tripanocidas/farmacologíaRESUMEN
Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.