RESUMO
Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus. Benzylamines are a class of compounds selectively designed to inhibit the squalene synthase (SQS) that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzylamines (SBC 37-43) against Leishmania amazonensis. After the first screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating high selectivity. Analysis of the sterol composition revealed a depletion of endogenous 24-alkylated sterols such as episterol and 5-dehydroepisterol, with a concomitant accumulation of fecosterol, implying a disturbance in cellular lipid content. This result suggests a blockade of de novo sterol synthesis at the level of SQS and C-5 desaturase. Furthermore, physiological analysis and electron microscopy revealed three main alterations: (1) in the mitochondrion; (2) the presence of lipid bodies and autophagosomes; and (3) the appearance of projections in the plasma membrane. In conclusion, our results support the notion that benzylamines have a potent effect against Leishmania amazonensis and should be an exciting novel pharmaceutical lead for developing new chemotherapeutic alternatives to treat leishmaniasis.
Assuntos
Leishmania mexicana , Leishmania , Benzilaminas/farmacologia , Farnesil-Difosfato Farnesiltransferase/metabolismo , Estresse Oxidativo , Esteróis/metabolismoRESUMO
Background: Osmanthus fragrans is an important ornamental tree and has been widely planted in China because of its pleasant aroma, which is mainly due to terpenes. The monoterpenoid and sesquiterpenoid metabolic pathways of sweet osmanthus have been well studied. However, these studies were mainly focused on volatile small molecule compounds. The molecular regulation mechanism of synthesis of large molecule compounds (triterpenoids) remains unclear. Squalene synthase (SQS), squalene epoxidase (SQE), and beta-amyrin synthase (BETA-AS) are three critical enzymes of the triterpenoid biosynthesis pathway. Results: In this study, the full-length cDNA and gDNA sequences of OfSQS, OfSQE, and OfBETA-AS were isolated from sweet osmanthus. Phylogenetic analysis suggested that OfSQS and OfSQE had the closest relationship with Sesamum indicum, and OfBETA-AS sequence shared the highest similarity of 99% with that of Olea europaea. The qRT-PCR analysis revealed that the three genes were highly expressed in flowers, especially OfSQE and OfBETA-AS, which were predominantly expressed in the flowers of both "Boye" and "Rixiang" cultivars, suggesting that they might play important roles in the accumulation of triterpenoids in flowers of O. fragrans. Furthermore, the expression of OfBETA-AS in the two cultivars was significantly different during all the five flowering stages; this suggested that OfBETA-AS may be the critical gene for the differences in the accumulation of triterpenoids. Conclusion: The evidence indicates that OfBETA-AS could be the key gene in the triterpenoid synthesis pathway, and it could also be used as a critical gene resource in the synthesis of essential oils by using bioengineered bacteria.
Assuntos
Triterpenos/metabolismo , Clonagem Molecular , Oleaceae/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Óleos Voláteis , Expressão Gênica , Reação em Cadeia da Polimerase , Oleaceae/enzimologia , Esqualeno Mono-Oxigenase/metabolismo , OdorantesRESUMO
Glioblastoma (GBM) is the most common primary brain tumor. Genetic mutations may reprogram the metabolism of neoplastic cells. Particularly, alterations in cholesterol and fatty acid biosynthetic pathways may favor biomass synthesis and resistance to therapy. Therefore, compounds that interfere with those pathways, such as phytol (PHY) and retinol (RET), may be appropriate for cytotoxic approaches. We tested the effect of PHY or RET on the viability of human GBM cell lines (U87MG, A172 and T98G). Since the compounds showed a dose-dependent cytotoxic effect, additional analyses were performed with IC50 values. Transcriptome analyses of A172 cells treated with PHY IC50 or RET IC50 revealed down-regulated genes involved in cholesterol and/or fatty acid biosynthetic pathways. Thus, we investigated the expression of proteins required for cholesterol and/or fatty acid synthesis after treating all lineages with PHY IC50 or RET IC50 and comparing them with controls. Sterol regulatory element-binding protein 1 (SREBP-1) expression was reduced by PHY in U87 and T98G cells. However, fatty acid synthase (FAS) protein expression, which is regulated by SREBP-1, was down-regulated in all lineages after both treatments. Moreover, farnesyl-diphosphate farnesyltransferase (FDFT1) levels, a protein associated with cholesterol synthesis, were reduced in all lineages by PHY and in U87MG and A172 cells by RET. Our results suggest that SREBP-1, FAS and FDFT1 are potential target(s) for future in vivo approaches against GBM and support the use of inhibitors of their synthesis, including PHY and RET, for such approaches.
Assuntos
Antineoplásicos/farmacologia , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Glioblastoma/tratamento farmacológico , Fitol/farmacologia , Vitamina A/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácido Graxo Sintases/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Ergosterol/antagonistas & inibidores , Itraconazol/farmacologia , Leishmania mexicana/efeitos dos fármacos , Piridinas/farmacologia , Quinuclidinas/farmacologia , Triazóis/farmacologia , Tripanossomicidas/farmacologia , Animais , Meios de Cultura/química , Sinergismo Farmacológico , Quimioterapia Combinada , Ergosterol/biossíntese , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Farnesil-Difosfato Farnesiltransferase/metabolismo , Humanos , Leishmania mexicana/isolamento & purificação , Leishmania mexicana/metabolismo , Leishmania mexicana/ultraestrutura , Leishmaniose Tegumentar Difusa/parasitologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Testes de Sensibilidade Parasitária , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Esterol 14-Desmetilase/metabolismoRESUMO
Copper is an essential micronutrient that functions as an enzymatic cofactor in a wide range of cellular processes. Although adequate Cu levels are essential for normal metabolism, excess Cu can be toxic to cells. Cellular responses to copper deficiency and overload involve changes in the expression of genes directly and indirectly involved in copper metabolism. However little is known on the effect of physiological copper concentration on gene expression changes. In the current study we aimed to establish whether the expression of genes encoding enzymes related to cholesterol (hmgcs1, hmgcr, fdft) and fatty acid biosynthesis and LDL receptor can be induced by an iso-physiological copper concentration. The iso-physiological copper concentration was determined as the bioavailable plasmatic copper in a healthy adult population. In doing so, two blood cell lines (Jurkat and THP-1) were exposed for 6 or 24 h to iso- or supraphysiological copper concentrations. Our results indicated that in cells exposed to an iso-physiological copper concentration the early induction of genes involved in lipid metabolism was not mediated by copper itself but by the modification of the cellular redox status. Thus our results contributed to understand the involvement of copper in the regulation of cholesterol metabolism under physiological conditions.
Assuntos
Colesterol/biossíntese , Cobre/farmacologia , Expressão Gênica/efeitos dos fármacos , Histidina/análogos & derivados , Compostos Organometálicos/farmacologia , RNA Mensageiro/genética , Colesterol/genética , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Histidina/farmacologia , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Células Jurkat , Metabolismo dos Lipídeos/efeitos dos fármacos , Oxirredução , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
We investigated the molecular basis of the activity of 4-phenoxyphenoxyethyl thiocyanate (WC-9) against Trypanosoma cruzi, the etiological agent of Chagas' disease. We found that growth inhibition of T. cruzi epimastigotes induced by this compound was associated with a reduction in the content of the parasite's endogenous sterols due to a specific blockade of their de novo synthesis at the level of squalene synthase.