RESUMO
Gold nanorods (GNRs) are increasingly being studied for diagnostic and therapeutic purposes. Green synthesis based methods with natural compounds as additives stand out as a hope in terms of better synthesis methodology, with advantages of producing potentially less toxic and, perhaps, biologically active GNRs due to influence of natural additives used during synthesis. Exploring green chemistry using different natural phenolic compounds, the present work reveals different in vitro activity of GNRs evaluated against different parasites that causes skin infectious diseases compared to GNRs produced by convencional seed mediated method. This approach brings advantages in producing active GNRs, with ease calling, less cytotoxic and with a better selectivity index (SI) than GNRs synthesized by conventional seed mediated synthesis, opening new possibilities for therapies. Natural compounds used in green syntheses were gallic acid (GA), resveratrol (RSV) and a purified fraction of the hydroalcoholic extract of Stryphnodendron obovatum. GNRs exhibited great activity against Leishmania braziliensis, and the dermatophytes Tricophyton rubrum, T. interdigitale and Microsporum gypseum. The anti-Leishmania and antidermatophytic activity of GNRs reinforce the applicability of GNRs in biomedical field and the influence of synthesis method in biological activity, showing benefits related to the seedless synthesis with natural compounds. In addition, these preliminary results indicate the possibility of exploring at maximum the physical and chemical properties of GNRs in addition to the biological activity itself, such as the development of topical antiparasitic formulations for association with phototherapy.
Assuntos
Ouro , Nanotubos , Ouro/química , Resveratrol , Ácido Gálico/farmacologia , AntiparasitáriosRESUMO
BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data. The antileishmanial activity of the isolated compounds was evaluated in intracellular amastigote forms of Leishmania (Viannia) braziliensis expressing firefly luciferase as reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian cell lines by MTT assay. FINDINGS Fractionation of the extract yielded seven compounds: 10-acetyl trichoderonic acid A (1), 6'-acetoxy-piliformic acid (2), 5',6'-dehydropiliformic acid (3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first time. Compounds 1, 2, and 5 were more active, with IC50 values of 21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and THP-1 cells. MAIN CONCLUSIONS N. pseudotrichia produces secondary metabolites that are more toxic to intracellular amastigote forms of L. (V.) braziliensis than to mammalian cells.
Assuntos
Caesalpinia/microbiologia , Leishmania braziliensis/efeitos dos fármacos , Nectria/química , Tripanossomicidas/farmacologia , Animais , Sobrevivência Celular , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária , Testes de Toxicidade , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/toxicidade , Células VeroRESUMO
Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N 6 -methyladenosine (m 6 A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to numerous cancers. Here we show that changes in m 6 A modification location can impact oncogenesis. A gain-of-function missense mutation found in cancer patients, METTL14 R298P , promotes malignant cell growth in culture and in transgenic mice. The mutant methyltransferase preferentially modifies noncanonical sites containing a GGAU motif and transforms gene expression without increasing global m 6 A levels in mRNAs. The altered substrate specificity is intrinsic to METTL3-METTL14, helping us to propose a structural model for how the METTL3-METTL14 complex selects the cognate RNA sequences for modification. Together, our work highlights that sequence-specific m 6 A deposition is important for proper function of the modification and that noncanonical methylation events can impact aberrant gene expression and oncogenesis.
RESUMO
The Leishmania aquaglyceroporin 1 (AQP1) plays an important role in osmoregulation and antimony (Sb) uptake, being determinant for resistance to antimony. We have previously demonstrated that G133D mutation on L. guyanensis AQP1 (LgAQP1) leads to reduced Sb uptake. Here, we investigated the effects of G133D mutation on LgAQP1 structure, associated with Sb uptake and alterations in osmoregulation capacity. High confidence molecular models of wild-type LgAQP1 as well as the LgAQP1::G133D mutant were constructed and optimized via comparative homology modeling. Computational methods from the mCSM platform were used to evaluate the effects on protein stability and on its ability to bind to glycerol. Functional validation of the disruptive effect of the mutation on LgAQP1 was done by challenging the parasites with hypo-osmotic chock. Glycine 133 is on transmembrane helix 3, buried in the membrane in both open and closed conformation. G133D mutation was predicted to be highly destabilizing, as it alters the helical bundling arrangement in order to accommodate the aspartic acid side chain. The shift in helices also resulted in fewer favorable contacts with glycerol in the channel, which would explain the reduced affinity for similar small molecules as SbO3. Under hypo-osmotic condition, L. guyanensis AQP1G133D presented a 3-fold increase in cellular volume and pronounced delay to recover osmosis homeostasis when compared to the wild-type, a profile that was enhanced in LgAQP1-/- mutants. In conclusion, G133D is a highly disruptive mutation that will destabilize the monomer, compromise tetramer formation and alter pore conformation, leading to reduced Sb uptake and deficient osmoregulation.
Assuntos
Aquaporina 1/genética , Leishmania guyanensis/genética , Mutação , Pressão Osmótica , Proteínas de Protozoários/genética , Animais , Aquaporina 1/química , Leishmania guyanensis/fisiologia , Modelos Moleculares , Proteínas de Protozoários/químicaRESUMO
Despite all efforts to provide new chemical entities to tackle leishmaniases, we are still dependent on a the limited drug arsenal, together with drawbacks like toxicity and drug-resistant parasites. Collaborative drug discovery emerged as an option to speed up the way to find alternative antileishmanial agents. This is the case of Medicines for Malaria Ventures - MMV, that promotes an open source drug discovery initiative to fight diseases worldwide. Here, we screened 400 compounds from 'Pathogen Box' (PBox) collection against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in Brazil. Twenty-three compounds were able to inhibit ≥ 80 % L. braziliensis growth at 5 µM. Six out of the PBox selected 23 compounds were found to be highly selective against L. braziliensis intracellular amastigotes with selectivity index varying from > 104 to > 746 and IC50s ranging from 47 to 480 nM. The compounds were also active against antimony-resistant L. braziliensis isolated from the field or laboratory selected mutants, revealing the potential on treating patients infected with drug resistant parasites. Most of the selected compounds were known to be active against kinetoplastids, however, two compounds (MMV688703 and MMV676477) were part of toxoplasmosis and tuberculosis 'PBox' disease set, reinforcing the potential of phenotyping screening to unveil drug repurposing. Here we applied a computational prediction of pharmacokinetic properties using the ADMET predictor pkCSM (http://biosig.unimelb.edu.au/pkcsm/). The tool offered clues on potential drug development needs and can support further in vivo studies. Molecular docking analysis identified CRK3 (LbrM.35.0660), CYP450 (LbrM.30.3580) and PKA (LbrM.18.1180) as L. braziliensis targets for MMV676604, MMV688372 and MMV688703, respectively. Compounds from 'Pathogen Box' thus represents a new hope for novel (or repurposed) small molecules source to tackle leishmaniases.
Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Descoberta de Drogas , Reposicionamento de Medicamentos , Resistência a Medicamentos , Leishmania braziliensis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Animais , Antiprotozoários/química , Antiprotozoários/farmacocinética , Antiprotozoários/toxicidade , Simulação por Computador , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Leishmania braziliensis/crescimento & desenvolvimento , Modelos Biológicos , Modelos Químicos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Células THP-1RESUMO
Four gold(I) complexes conceived as anticancer agents were synthesized by reacting [Au(PEt3 )Cl] and [Au(PPh3 )Cl] with ligands derived from δ-d-gluconolactone. The ligands' structure was designed to combine desired biological properties previously reported for each group. Ligands were synthesized from δ-d-gluconolactone via ketal protection and hydrazide formation followed by cyclization with CS2 to produce the novel oxadiazolidine-2-thione 7 and 8. Increasing of the ligands' lipophilicity via ketal protection proved useful since all four gold(I) complexes showed anticancer and antileishmanial properties. The IC50 values are at low micromolar range, varying from 2 to 3 µm for the most active compounds. The free D-gluconate 1,3,4 oxadiazole-derived ligands were neither toxic nor presented anticancer or antileishmanial properties. Triethylphosphine-derived compounds 9 and 10 were more selective against B16-F10 melanoma cell line. Although similar in vitro antileishmanial activity was observed for the gold(I) precursors themselves and their derived complexes, the latter were three times less toxic for human THP-1 macrophage cell line; this result is attributed to an isomeric variation of the D-gluconate ligand and the oxadiazole portion, which was one of the key concepts behind this work. These findings should encourage further research on gold(I) complexes to develop novel compounds with potential application in cancer and leishmaniasis chemotherapy.
Assuntos
Antineoplásicos/química , Antiprotozoários/química , Complexos de Coordenação/química , Gluconatos/química , Ouro/química , Lactonas/química , Tionas/química , Animais , Antineoplásicos/farmacologia , Antiprotozoários/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/farmacologia , Ciclização , Humanos , Leishmania/efeitos dos fármacos , Ligantes , Camundongos , Oxidiazóis/química , Relação Estrutura-AtividadeRESUMO
Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. Only two drugs are available, with the drawback of low rate of cure in the chronic phase of the disease and undesirable side effects. These facts highlight the need to find new compounds for Chagas disease chemotherapy. We describe the isolation and identification of an inseparable mixture of two new trixikingolides from Trixis vauthieri, a plant from family Asteraceae, which present outstanding in vitro trypanocidal activity, with IC50 value of 0.053 µM against the intracellular trypomastigotes and amastigotes forms of T. cruzi infecting L929 cells. The IC50 of the mixture against the host cells is 68 times higher and about 70 times more potent than benznidazole, the reference drug used as control at the experiments. The next step, which depends on obtaining larger quantities of the mixture, is to test it on mice infected with T. cruzi.
Assuntos
Asteraceae , Doença de Chagas , Tripanossomicidas , Trypanosoma cruzi , Animais , Asteraceae/química , Doença de Chagas/tratamento farmacológico , Camundongos , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The drugs currently used to treat leishmaniases have limitations concerning cost, efficacy, and safety, making the search for new therapeutic approaches urgent. We found that the gold(I)-derived complexes were active against L. infantum and L. braziliensis intracellular amastigotes with IC50 values ranging from 0.5 to 5.5 µM. All gold(I) complexes were potent inhibitors of trypanothione reductase (TR), with enzyme IC50 values ranging from 1 to 7.8 µM. Triethylphosphine-derived complexes enhanced reactive oxygen species (ROS) production and decreased mitochondrial respiration after 2 h of exposure, indicating that gold(I) complexes cause oxidative stress by direct ROS production, by causing mitochondrial damage or by impairing TR activity and thus accumulating ROS. There was no cross-resistance to antimony; in fact, SbR (antimony-resistant mutants) strains were hypersensitive to some of the complexes. BALB/c mice infected with luciferase-expressing L. braziliensis or L. amazonensis and treated orally with 12.5 mg/kg/day of AdT Et (3) or AdO Et (4) presented reduced lesion size and parasite burden, as revealed by bioimaging. The combination of (3) and miltefosine allowed for a 50% reduction in miltefosine treatment time. Complexes 3 and 4 presented favorable pharmacokinetic and toxicity profiles that encourage further drug development studies. Gold(I) complexes are promising antileishmanial agents, with a potential for therapeutic use, including in leishmaniasis caused by antimony-resistant parasites.
Assuntos
Antiprotozoários/farmacologia , Ouro/farmacologia , Leishmaniose , NADH NADPH Oxirredutases/antagonistas & inibidores , Animais , Leishmaniose/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Estresse OxidativoRESUMO
Gold(I)-containing complexes are used in drug discovery research for rheumatoid arthritis, cancer, and parasitic infections. In this study, we tested the bioactivity of gold(I) complexes in vivo using planarians. The planarian Schmidtea mediterranea possesses orthologues of tumor suppressor genes, such as p53, that, when silenced, cause deregulation of cell proliferation and apoptosis. In this context, we tested two triethylphosphine-gold(I) complexes (AdO and AdT) to determine if they can attenuate phenotypes that result from p53 inhibition. First, we identified the drug concentration that did not affect survival or regeneration and evaluated the drug's effect on cell division and apoptosis. We found that AdT treatment decreased the number of mitotic cells and that all drug treatments increased the number of apoptotic cells. We then performed p53(RNAi) and drug treatments concomitantly and observed the phenotype progression. Drug treatment increased survival three-fold and decreased apoptosis, which resulted in an attenuated phenotype. Our results indicate that planarians can be treated with gold(I) complexes, and that this treatment can diminish the p53(RNAi) phenotype and extend survival. In this work we show that planarians can be used as a model to study the in vivo effect of gold(I) complexes and to further investigate their mechanisms of action.
Assuntos
Complexos de Coordenação/química , Tiossulfato Sódico de Ouro/química , Ouro/química , Planárias/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Complexos de Coordenação/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Ouro/farmacologia , Tiossulfato Sódico de Ouro/farmacologia , Humanos , Planárias/genética , Interferência de RNA/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.
Assuntos
Citocinese , Leishmania infantum , Leishmaniose Visceral , Mutação , Animais , Citocinese/genética , Citocinese/imunologia , Modelos Animais de Doenças , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/imunologia , Humanos , Leishmania infantum/genética , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/imunologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/prevenção & controle , Pontos de Checagem da Fase M do Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Plasmídeos/genética , Plasmídeos/imunologia , Plasmídeos/metabolismo , Células THP-1RESUMO
The current anticancer and antileishmanial drug arsenal presents several limitations concerning their specificity, efficacy, costs and the emergence of drug-resistant cells lines, which encourages the urgent need to search for new alternatives. Inspired by the fact that gold(I)-based compounds are promising antitumoral and antileishmanial drug candidates, we synthesized novel gold(I) complexes containing phosphine and 5-phenyl-1,3,4-oxadiazole-2-thione and evaluated their anticancer and antileishmanial activities. Synthesis was performed by reacting 5-phenyl-1,3,4-oxadiazole-2-thione derivatives with chloro(triphenylphosphine)gold(I) and chloro(triethylphosphine)gold(I). The novel compounds were characterized by infrared, Raman, 1H, 13C nuclear magnetic resonance, high-resolution mass spectra, and x-ray crystallography. The coordination of the ligands to gold(I) occurred through the exocyclic sulfur atom. All gold(I) complexes were active at low micromolar or nanomolar range with IC50 values ranging from <0.10 to 1.66 µM against cancer cell lines and from 0.9 to 4.2 µM for Leishmania infantum intracellular amastigotes. Compound (6-A) was very selective against murine melanoma B16F10, colon cancer CT26.WT cell lines and L. infantum intracellular amastigotes. Compound (7-B) presented the highest anticancer activity against both cancer cell lines while the promising antileishmanial lead was compound (6-A). Tiethylphosphine gold(I) complexes were more active than the conterparts triphenylphosphine derivatives for both anticancer and antileishmanial activities. Triethylphosphine gold(I) derivatives presented antimony cross-resistance in L. guyanensis demonstrating their potential to be used as chemical tools to better understand mechanisms of drug resistance and action. These findings revealed the anticancer and antileishmanial potential of gold(I) oxadiazole phosphine derivatives.
Assuntos
Desenho de Fármacos , Ouro/química , Leishmania infantum/efeitos dos fármacos , Compostos Organoáuricos/química , Compostos Organoáuricos/farmacologia , Oxidiazóis/química , Fosfinas/química , Antimônio/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular Tumoral , Resistência a Medicamentos/efeitos dos fármacos , HumanosRESUMO
BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data. The antileishmanial activity of the isolated compounds was evaluated in intracellular amastigote forms of Leishmania (Viannia) braziliensis expressing firefly luciferase as reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian cell lines by MTT assay. FINDINGS Fractionation of the extract yielded seven compounds: 10-acetyl trichoderonic acid A (1), 6′-acetoxy-piliformic acid (2), 5′,6′-dehydropiliformic acid (3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first time. Compounds 1, 2, and 5 were more active, with IC50 values of 21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and THP-1 cells. MAIN CONCLUSIONS N. pseudotrichia produces secondary metabolites that are more toxic to intracellular amastigote forms of L. (V.) braziliensis than to mammalian cells.
Assuntos
Leishmania braziliensis/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Testes de Toxicidade , Caesalpinia/microbiologia , Sobrevivência Celular , Chlorocebus aethiops , Concentração Inibidora 50RESUMO
As leishmanioses e o câncer são doenças complexas, com amplo espectro de manifestações clínicas e que dependem da quimioterapia para seu controle. As opções de tratamento para essas doenças são limitadas e possuem diversas desvantagens como alto custo e toxicidade, fazendo com que a busca por novos fármacos seja prioridade. Neste trabalho investigamos a atividade e modo de ação de complexos de ouro (AuI) que foram sintetizados com base na auranofina, um fármaco que tem atividades antileishmania e antitumoral descritas. Com o objetivo de implementar um modelo in vivo de baixo custo e fácil manutenção para o estudo de vias supressoras de tumores e compostos antitumorais, usamos planárias da espécie Schmidtea mediterranea. Os 24 complexos de ouro(I) estudados possuem potente atividades antileishmania e antitumoral, com CI50 entre 0,5 e 4,2 µM em amastigotas de Leishmania infantum e 0,1 e 5,7 µM em linhagem celular derivada de melanoma murino. Os compostos AdO Et e AdT Et causam estresse oxidativo nos parasitos, gerando espécies reativas de oxigênio diretamente ou causando seu acúmulo por inibição da enzima tripanotiona redutase e dano mitocondrial. Estes complexos foram capazes de reduzir o tamanho de lesão e carga parasitária em modelo murino de leishmaniose cutânea. Além disso, a combinação de AdT Et e miltefosina foi capaz de reduzir o tempo de tratamento pela metade. O estudo destes complexos utilizando planárias nos permitiu observar que eles causam alterações no ciclo celular, reduzindo taxas de mitose e aumentando taxas de apoptose em animais saudáveis. Em planárias cuja P53 foi silenciada, observamos que os compostos AdO Et e AdT Et aumentaram a sobrevida dos animais, por agirem inibindo a via de apoptose independente de P53. Os resultados deste trabalho indicam que os complexos de ouro(I) estudados são promissores agentes antileishmania e antitumorais. Sua comprovada eficácia in vivo, aliado a parâmetros toxicológicos e farmacocinéticos favoráveis, encorajam o desenvolvimento de alternativas para tratamento das leishmanioses e câncer, contribuindo com a expansão do restrito arsenal terapêutico.