RESUMO
Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.
RESUMO
Animal toxins present high selectivity and specificity for their molecular targets, and have long been considered as prototypes for developing novel drugs, with some successful cases. In this regard, the variety of molecules found in animal venoms, which can be capable of affecting vital physiological systems, have providing the development of studies focusing on turning those molecules (toxins) into therapeutics to treat several diseases, such as chronic pain, hypertension, thrombosis, cancer, and so on. However, some important issues have been responsible for disrupting the toxin-based drug discovery projects. In this review, we have briefly highlighted the development of drugs based on animal toxins, discussing some successful cases as well as the main causes of failure, pointing out the recent strategies applied to overcome the difficulties related to the translational process in this kind of development scenario.
Assuntos
Descoberta de Drogas , Peptídeos , Toxinas Biológicas , Peçonhas , Animais , Dor Crônica/tratamento farmacológico , Humanos , Hipertensão/tratamento farmacológico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Peptídeos/efeitos adversos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Trombose/tratamento farmacológico , Toxinas Biológicas/química , Toxinas Biológicas/farmacologia , Peçonhas/química , Peçonhas/farmacologiaRESUMO
Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 µL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and ß3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.
Assuntos
Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas e Peptídeos Salivares/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Artrópodes , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dinoprostona/metabolismo , Células Endoteliais/metabolismo , Biblioteca Gênica , Masculino , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas e Peptídeos Salivares/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.
Assuntos
Animais , Masculino , Coelhos , Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Hemorragia/tratamento farmacológico , Antagonistas de Heparina/farmacologia , Heparina de Baixo Peso Molecular/antagonistas & inibidores , Protrombina/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Hemorragia/induzido quimicamenteRESUMO
BACKGROUND: Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells. METHODS: The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT-PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells. RESULTS: B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA(121), (165), (189,) and (165b) in SK-MEL-25 and SCC-9 cells. CONCLUSION: FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintases/antagonistas & inibidores , Lactonas/farmacologia , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/secundário , Melanoma/irrigação sanguínea , Melanoma/secundário , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Bucais/tratamento farmacológico , Orlistate , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacosRESUMO
Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.
Assuntos
Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Hemorragia/tratamento farmacológico , Antagonistas de Heparina/farmacologia , Heparina de Baixo Peso Molecular/antagonistas & inibidores , Protrombina/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Animais , Hemorragia/induzido quimicamente , Masculino , CoelhosRESUMO
Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 µl; each 48 h) inhibited VEGF-A-induced (10 ng/10 µl; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas e Peptídeos Salivares/farmacologia , Inibidores de Serina Proteinase/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Células Endoteliais/metabolismo , Inibidores do Fator Xa , Ixodidae/metabolismo , Masculino , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Tela Subcutânea/efeitos dos fármacos , Tela Subcutânea/metabolismoRESUMO
Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.
Assuntos
Ceratite por Acanthamoeba/patologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/enzimologia , Proteínas de Protozoários/metabolismo , Serina Proteases/metabolismo , Acanthamoeba/isolamento & purificação , Adulto , Eletroforese , Humanos , Pessoa de Meia-Idade , Peso Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Índice de Gravidade de Doença , Estatística como Assunto , Especificidade por SubstratoRESUMO
Despite the nearly worldwide distribution of Lepidoptera, there are few species with clear documentation of adverse reactions in humans. Most syndromes caused by Lepidoptera are consequences of direct contact with the hairs or setae of caterpillars. In most instances, the adverse effects caused by moth and caterpillars are self-limited and the treatment is based on the removal of hairs, application of topical antipruritics and, in some cases, the use of oral antihistamines. However, in the case of envenoming by South American Lonomiaobliqua caterpillars, the antilonomic serum produced at Instituto Butantan in Brazil is the only effective treatment to re-establish the physiological coagulation parameters in poisoned patients and to abolish the complications seen in severe cases (e.g. consumptive coagulopathy, intracerebral hemorrhage, and acute renal failure). Many studies have been carried out to understand the pathophysiological mechanism of envenoming by L. obliqua. Several toxic principles were found in bristle extract and the hemolymph, probably related to the envenoming. An interesting fact is that some toxins from the venom usually have more than one function. With the advent of molecular biology techniques it has become possible to analyze these processes at a molecular level, thus giving rise to hypotheses on the molecular basis of envenomation. This review contributes to enhance our understanding of the dramatic alterations that hemorrhagic syndrome causes in patients, current treatment, and the diversity of the molecules involved in this pathology.
Assuntos
Venenos de Artrópodes/toxicidade , Coagulação Sanguínea/efeitos dos fármacos , Hemorragia/etiologia , Mariposas/química , Serina Endopeptidases/toxicidade , Animais , Venenos de Artrópodes/química , Humanos , Larva/química , Serina Endopeptidases/químicaRESUMO
Bioactive compounds of great interest are found in the saliva of hematophagous organisms. While exploring a cDNA library derived from the salivary glands of the tick Amblyomma cajennense, a transcript that codes for a protein with unique structure (containing an N-terminal Kunitz-type domain and a C-terminus with no homology to any annotated sequences) was found. The recombinant mature form of this protein ( approximately 13.5kDa) was produced in Escherichia coli BL21 (DE3), and it was able to inhibit Factor Xa (FXa) and extend global blood clotting times in vitro and ex vivo. Static and dynamic predictions of its tertiary structure indicate regions that may be related to its FXa inhibitor function.