RESUMO
Polymer polyethylene glycol (PEG), or PEGylation of polypeptides improves protein drug stability by decrease degradation and reduces renal clearance. To produce a pharmaceutical disintegrin derivative, the N-terminal PEGylation technique was used to modify the disintegrin derivative [KGDRR]trimucrin for favorable safety and pharmacokinetic profiles and antithrombotic efficacy. We compared intact [KGDRR]trimucrin (RR) and PEGylated KGDRR (PEG-RR) by in vitro and in vivo systems for their antithrombotic activities. The activity of platelet aggregation inhibition and the bleeding tendency side effect were also investigated. PEG-RR exhibited optimal potency in inhibiting platelet aggregation of human/mouse platelet-rich plasma activated by collagen or ADP with a lower IC50 than the intact derivative RR. In the illumination-induced mesenteric venous thrombosis model, RR and PEG-RR efficaciously prevented occlusive thrombosis in a dose-dependent manner. In rotational thromboelastometry assay, there was no effect of PEG-RR in human whole blood coagulation even given at a higher concentration (30 µg/mL), while RR slightly prolonged clotting time. However, RR and PEG-RR were not associated with severe thrombocytopenia or bleeding in FcγRIIa-transgenic mice at equally efficacious antithrombotic dosages. We also found the in vivo half-life of PEGylation was longer than RR (RR: 15.65 h vs. PEG-RR: 20.45 h). In conclusion, injectable PEG-RR with prolonged half-life and decreased bleeding risk is a safer anti-thrombotic agent for long-acting treatment of thrombus diseases.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Desintegrinas/farmacologia , Fibrinolíticos/farmacologia , Peptídeos/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Polietilenoglicóis/química , Trombose/prevenção & controle , Animais , Modelos Animais de Doenças , Desintegrinas/química , Desintegrinas/toxicidade , Composição de Medicamentos , Fibrinolíticos/química , Fibrinolíticos/toxicidade , Hemorragia/induzido quimicamente , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Peptídeos/química , Peptídeos/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Trombocitopenia/induzido quimicamente , Trombose/sangue , Trombose/etiologiaRESUMO
Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.
Assuntos
Cisteína Endopeptidases/genética , Proteína SUMO-1/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/toxicidade , Plaquetas/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/toxicidade , Cisteína Endopeptidases/metabolismo , Desintegrinas/genética , Desintegrinas/toxicidade , Escherichia coli/genética , Humanos , Fosfolipase D/genética , Fosfolipase D/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Proteína SUMO-1/metabolismo , Venenos de Aranha , AranhasRESUMO
We have demonstrated in previous studies that a single amino acid change can alter the activity of the recombinant disintegrin r-Moj. In this study, four r-Moj recombinants containing single mutations (r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-MN) and two containing double mutations (r-Moj-MP and r-Moj-NM) at the binding loop were produced, purified, and tested. All r-Moj-W_, r-Moj-M_, and r-Moj-NM mutant peptides inhibited platelet aggregation at higher potency than r-Moj-D_ mutants. Five of the seven r-Moj peptides inhibited angiogenesis at different levels. Two of the mutant peptides with a methionine at the second position carboxyl of the RGD (r-Moj-WM and r-Moj-NM) were the strongest angiogenesis inhibitors, with r-Moj-WM being the most potent. Recombinant r-Moj-MP and r-Moj-WN failed to inhibit angiogenesis. Only the r-Moj-MP mutant peptide induced apoptosis of SK-Mel-28 cells significantly (p = 0.001). This was confirmed by chromatin condensation. Proliferation of SK-Mel-28 cells was inhibited at high levels (>70%) by all r-Moj mutant peptides. Recombinant r-Moj-MN and r-Moj-WN failed to inhibit cell migration significantly (p > 0.5). Recombinant r-Moj-NM was the strongest cell migration inhibitor (98% ± 0.69), followed by r-Moj-MP (80% ± 2.87), and r-Moj-WM (61.8% ± 5.45). The lowest inhibitor was r-Moj-WL (50% ± 12.16). Our functional data suggest that the most potent r-Moj disintegrins contain a methionine in the first or the second position carboxyl to the RGD.
Assuntos
Desintegrinas/toxicidade , Metionina/metabolismo , Mutação , Proteínas Recombinantes/toxicidade , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Desintegrinas/química , Desintegrinas/genética , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
RNA sequencing of untreated and r-Moj-DM treated SK-Mel-28 cells was performed after 6 h, to begin unraveling the apoptotic pathway induced by r-Moj-DM. Bioinformatic analyses of RNA sequencing data yielded 40 genes that were differentially expressed. Nine genes were upregulated and 31 were downregulated. qRT-PCR was used to validate differential expression of 13 genes with known survival or apoptotic-inducing activities. Expression of BNiP3, IGFBP3, PTPSF, Prune 2, TGF-ß, and TXNIP were compared from cells treated with r-Moj-DN (a strong apoptotic inducer) or r-Moj-DA (a non-apoptotic inducer) for 1 h, 2 h, 4 h, and 6 h after treatment. Our results demonstrate that significant differences in expression are only detected after 4 h of treatment. In addition, expression of TXNIP (an apoptotic inducer) remains elevated at 4 h and 6 h only in r-Moj-DN treated cells. Based on the consistency of elevated TXNIP expression, we further studied TXNIP as a novel target of disintegrin activation. Confocal microscopy of anti-TXNIP stained SK-Mel-28 cells suggests nuclear localization of TXNIP after r-Moj-DM treatment. A stable TXNIP knockdown SK-Mel-28 cell line was produced to test TXNIP' role in the apoptotic induction by r-Moj-DM. High cell viability (74.3% ±9.1) was obtained after r-Moj-DM treatment of TXNIP knocked down SK-Mel-28 cells, compared to 34% ±0.187 for untransduced cells. These results suggest that TXNIP is required early in the apoptotic-inducing pathway resulting from r-Moj-DM binding to the αv integrin subunit.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/fisiologia , Desintegrinas/toxicidade , Análise de Sequência de RNA , Apoptose/fisiologia , Proteínas de Transporte/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , HumanosRESUMO
Melittin, which acts as a membrane-disrupting lytic peptide, is not only cytotoxic to tumors, but also vital to normal cells. Melittin had low toxicity when coupled with target peptides. Despite significant research development with the fused toxin, a new fused toxin is needed which has a cleavable linker such that the fused toxin can release melittin after protease cleavage on the tumor cell surface. We describe a novel fused toxin, composed of disintegrin, uPA (urokinase-type plasminogen activator)-cleavable linker, and melittin. Disintegrin is a single strand peptide (73 aa) isolated from Gloydius Ussuriensis venom. The RGD (Arg-Gly-Asp) site of disintegrin dominates its interaction with integrins on the surface of the tumor cells. uPA is over-expressed and plays an important role in tumor cell invasiveness and metastatic progression. The DLM (disintegrin-linker-melittin) linker is uPA-cleavable, enabling DLM to release melittin. We compared binding activity of our synthesized disintegrin with native disintegrin and report that DLM had less binding activity than the native form. uPA-cleavage was evaluated in vitro and the uPA-cleavable linker released melittin. Treating tumors expressing uPA with DLM enhanced tumor cell killing as well as reduced toxicity to erythrocytes and other non-cancerous normal cells. The mechanism behind DLM tumor cell killing was tested using a DNA ladder assay, fluorescent microscopy, flow cytometry, and transmission electron microscopy. Data revealed tumor cell necrosis as the mechanism of cell death, and the fused DLM toxin with an uPA-cleavable linker enhanced tumor selectivity and killing ability.
Assuntos
Antineoplásicos/farmacologia , Desintegrinas/farmacologia , Meliteno/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Antineoplásicos/química , Antineoplásicos/toxicidade , Plaquetas/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desintegrinas/genética , Desintegrinas/metabolismo , Desintegrinas/toxicidade , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos , Meliteno/genética , Meliteno/metabolismo , Meliteno/toxicidade , Microscopia de Fluorescência , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Disintegrins, a family of polypeptides released in the venoms of viperid snakes (vipers and rattlesnakes) by the proteolytic processing of multidomain metalloproteinases, selectively block the function of ß(1) and ß(3) integrin receptors. Few of the proteins isolated and characterized from snake venoms have proven to be more structural and functional versatile than the disintegrins. Not surprisingly, 25 years after their discovery, our knowledge on the evolutionary history and the molecular determinants modulating the integrin inhibitory activity of disintegrins still remain fragmentary. This paper highlights some seminal contributions, including personal accounts of pioneer authors, related to basic and applied research on disintegrins. Investigators have evaluated disintegrin applications in therapies for a number of pathologies in which integrin receptors play relevant roles, particularly myocardial infarction and inappropriate tumor angiogenesis. Completing the continuing story of the disintegrin family by applying novel research approaches may hold the key to learn how to use deadly toxins as therapeutic agents.
Assuntos
Desintegrinas/toxicidade , Venenos de Serpentes/química , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Sequência Conservada , Desintegrinas/química , Desintegrinas/isolamento & purificação , Desintegrinas/farmacologia , Evolução Molecular , Humanos , Neoplasias/tratamento farmacológico , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Cell migration is a key process for the defense of pluricellular organisms against pathogens, and it involves a set of surface receptors acting in an ordered fashion to contribute directionality to the movement. Among these receptors are the integrins, which connect the cell cytoskeleton to the extracellular matrix components, thus playing a central role in cell migration. Integrin clustering at focal adhesions drives actin polymerization along the cell leading edge, resulting in polarity of cell movement. Therefore, small integrin-binding proteins such as the snake venom disintegrins that inhibit integrin-mediated cell adhesion are expected to inhibit cell migration. Here we review the current knowledge on disintegrin and disintegrin-like protein effects on cell migration and their potential use as pharmacological tools in anti-inflammatory therapy as well as in inhibition of metastatic invasion.
Assuntos
Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Desintegrinas/toxicidade , Neutrófilos/efeitos dos fármacos , Venenos de Serpentes/química , Animais , Anoikis/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Desintegrinas/antagonistas & inibidores , Humanos , Metástase Neoplásica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Venenos de Serpentes/farmacologiaRESUMO
The isolation and study of individual snake venom components paves the way for a deeper understanding of the pathophysiology of envenomings--thus potentially contributing to improved therapeutic modalities in the clinical setting--and also opens possibilities for the discovery of novel toxins that might be useful as tools for dissecting cellular and molecular processes of biomedical importance. This review provides a summary of the different toxins that have been isolated and characterized from the venom of Bothrops asper, the snake species causing the majority of human envenomings in Central America. This venom contains proteins belonging to at least eight families: metalloproteinase, serine proteinase, C-type lectin-like, L-amino acid oxidase, disintegrin, DC-fragment, cystein-rich secretory protein, and phospholipase A(2). Some 25 venom proteins within these families have been isolated and characterized. Their main biochemical properties and toxic actions are described, including, in some cases, their possible relationships to the pathologic effects induced by B. asper venom.
Assuntos
Bothrops , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/toxicidade , Animais , Venenos de Crotalídeos/enzimologia , Desintegrinas/química , Desintegrinas/metabolismo , Desintegrinas/toxicidade , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/metabolismo , L-Aminoácido Oxidase/toxicidade , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Metaloproteases/química , Metaloproteases/metabolismo , Metaloproteases/toxicidade , Modelos Moleculares , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Fosfolipases A2/toxicidade , Serina Proteases/química , Serina Proteases/metabolismo , Serina Proteases/toxicidadeRESUMO
To examine the toxicological effect of saxatilin, a disintegrin isolated from the venom of a Korean snake (Gloydius saxatilis), recombinant saxatilin was highly expressed as a biologically active form in Pichia pastoris, and was successfully purified to homogeneity from the culture broth supernatant. The molecular and biological properties of the recombinant protein were the same as those of its natural form. Plasma half-life of the protein in rat was determined to 13.8 min. The maximum tolerated dose of the recombinant saxatilin was examined in ICR mice. The determined LD(50) values were 400 and 600 mg/kg of the body weight of a male and female mouse, respectively. To investigate the repeated dose toxicity of saxatilin in mice, the test item was intravenously administered to groups of ICR mice every day for 4 weeks. We observed a decrease in locomotor activity, piloerection, and crouching in clinical findings, a decrease of red blood cells (RBCs) in hematology, and hyperplasia of the spleen in histology related to administration of the test item. These results suggest that the target organ of intravenous administration of the test item is the spleen. The no adverse effect level (NOAEL) in this test for both males and females is considered to be 3mg/kg. Our results also indicate that recombinant saxatilin is non-toxic at an administration dose with an anti-platelet effect, and might be a potential anti-adhesion therapeutic agent for thrombosis, cancer, restenosis, cataract, and osteoporosis.
Assuntos
Desintegrinas/administração & dosagem , Desintegrinas/toxicidade , Viperidae , Animais , Desintegrinas/química , Desintegrinas/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Tamanho do Órgão/efeitos dos fármacos , Organismos Geneticamente Modificados , Pichia/genética , Pichia/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Baço/efeitos dos fármacos , Baço/patologia , Urina , Redução de Peso/efeitos dos fármacosRESUMO
Disintegrins constitute a family of potent polypeptide inhibitors of integrins. Integrins are transmembrane heterodimeric molecules involved in cell-cell and cell-extracellular matrix interactions. They are involved in many diseases such as cancer and thrombosis. Thus, disintegrins have a great potential as anticancer and antithrombotic agents. A novel heterodimeric disintegrin was isolated from the venom of saw-scaled viper (Echis carinatus) and was crystallized. The crystals diffracted to 1.9 A resolution and belonged to space group P4(3)2(1)2. The data indicated the presence of a pseudosymmetry. The structure was solved by applying origin shifts to the disintegrin homodimer schistatin solved in space group I4(1)22 with similar cell dimensions. The structure refined to the final R(cryst)/R(free) factors of 0.213/0.253. The notable differences are observed between the loops, (Gln39-Asp48) containing the important Arg42-Gly43-Asp44, of the present heterodimer and schistatin. These differences are presumably due to the presence of two glycines at positions 43 and 46 that allow the molecule to adopt variable conformations. A comparative analysis of the surface-charge distributions of various disintegrins showed that the charge distribution on monomeric disintegrins occurred uniformly over the whole surface of the molecule, while in the dimeric disintegrins, the charge is distributed only on one face. Such a feature may be important in the binding of two integrins to a single dimeric disintegrin. The phylogenetic analysis developed on the basis of amino acid sequence and three-dimensional structures indicates that the protein diversification and evolution presumably took place from the medium disintegrins and both the dimeric and short disintegrins evolved from them.
Assuntos
Antineoplásicos/química , Desintegrinas/química , Inibidores da Agregação Plaquetária/química , Venenos de Víboras/química , Viperidae/metabolismo , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/toxicidade , Comunicação Celular , Cristalografia por Raios X , Dimerização , Desintegrinas/isolamento & purificação , Desintegrinas/toxicidade , Matriz Extracelular/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Filogenia , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/toxicidade , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/toxicidadeRESUMO
To explain the myotoxic effects of snake venoms, we searched for inhibitors of alpha7beta1 integrin, the major laminin-binding integrin in skeletal muscle. We discovered two inhibitors in the venom of Vipera lebetina. One of them, lebein-1 (known as lebein), has already been proposed to be a disintegrin because of its RGD-containing primary sequence. The other, lebein-2, is a novel protein that also interacts firmly with alpha3beta1, alpha6beta1, and alpha7beta1 integrins, but not with the collagen-binding alpha1beta1 and alpha2beta1 integrins. Ligand binding of laminin-recognizing beta1 integrins was efficiently blocked by both lebein-1 and lebein-2. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, neither lebein-1 nor lebein-2 interacted with alpha7beta1 integrin in an RGD-dependent manner, similar to the interaction of the laminin with alpha7beta1 integrin. Identical divalent cation dependence of integrin binding to laminin and to either of the two inhibitors and their mutually exclusive binding suggest that both lebein-1 and lebein-2 interact with the ligand-binding site of laminin-binding beta1 integrins by mimicking the yet unknown integrin-binding structure of laminins. Like lebein-1, lebein-2 is a soluble heterodimeric disintegrin of low molecular mass. Together with membrane-bound ADAM-2 and ADAM-9, the two inhibitors seem to form a small group of disintegrins that can bind to laminin-binding beta1 integrins. Because of their inhibitory capability both in vitro and in vivo, lebein-1 and lebein-2 may be valuable tools in influencing laminin-induced, integrin-mediated cell functions such as cell anchorage, migration, and mechanical force transduction on laminin-rich basement membranes.
Assuntos
Desintegrinas/química , Desintegrinas/toxicidade , Integrina beta1/efeitos dos fármacos , Integrina beta1/metabolismo , Laminina/metabolismo , Venenos de Víboras/química , Venenos de Víboras/toxicidade , Animais , Adesão Celular/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Venenos Elapídicos/toxicidade , Fibronectinas/metabolismo , Técnicas In Vitro , Integrina alfa3beta1/efeitos dos fármacos , Integrina alfa3beta1/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Cinética , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Oligopeptídeos/química , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.