RESUMO
In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Bauhinia/química , Neoplasias/tratamento farmacológico , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/farmacologia , Sementes/química , Catepsina G/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fluoruracila/farmacologia , Humanos , Neoplasias/patologia , Calicreína Plasmática/antagonistas & inibidores , Proteínas de Protozoários , Proteínas Recombinantes/genéticaRESUMO
Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.
Assuntos
Fabaceae/química , Inibidores de Proteases/farmacologia , Animais , Quimotripsina/antagonistas & inibidores , Fabaceae/classificação , Humanos , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Calicreína Plasmática/antagonistas & inibidores , Inibidores de Proteases/isolamento & purificação , Sementes/química , Sementes/classificação , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificação , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologiaRESUMO
Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.
Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.
Assuntos
Animais , Humanos , Fabaceae/química , Inibidores de Proteases/farmacologia , Quimotripsina/antagonistas & inibidores , Fabaceae/classificação , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Calicreína Plasmática/antagonistas & inibidores , Inibidores de Proteases/isolamento & purificação , Sementes/química , Sementes/classificação , Inibidor da Tripsina de Soja de Bowman-Birk/isolamento & purificação , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologiaRESUMO
Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 degrees C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI-I, CmPI-II and CmPI-III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI-I and CmPI-II was confirmed, while CmPI-III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI-I and CmPI-II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI-I (6 amino acids) and CmPI-II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI-II. Both inhibitors, CmPI-I and CmPI-II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (Ki) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI-I and CmPI-II are the first human neutrophil elastase inhibitors described in a mollusk.
Assuntos
Elastase de Leucócito/antagonistas & inibidores , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores de Serina Proteinase/farmacologia , Caramujos/química , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Oceanos e Mares , Elastase Pancreática/antagonistas & inibidores , Calicreína Plasmática/antagonistas & inibidores , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trombina/antagonistas & inibidores , Inibidores da Tripsina/farmacologiaRESUMO
The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.
Assuntos
Inibidores Enzimáticos/farmacologia , Heparina/farmacologia , Peptídeos/metabolismo , Calicreína Plasmática/antagonistas & inibidores , Calicreína Plasmática/metabolismo , Antitrombinas/farmacologia , Catálise , Proteína Inibidora do Complemento C1/farmacologia , Fator XII/efeitos dos fármacos , Fator XII/metabolismo , Humanos , Hidrólise , Peptídeos/efeitos dos fármacos , Calicreína Plasmática/química , Plasminogênio/efeitos dos fármacos , Plasminogênio/metabolismo , Estrutura Secundária de Proteína , Fatores de Tempo , alfa 1-Antitripsina/farmacologiaRESUMO
Bradykinin and Lys-bradykinin are potent peptide mediators implicated in several physiopathological effects in mammals. They act through activation of G-protein-coupled constitutive B(2) or inducible kinin B(1) receptors linked to signaling pathways involving increased intracellular Ca(++) concentrations and/or release of mediators including arachidonic acid metabolites, NO and EDHF. In the cardiovascular system, the kallikrein-kinin system exerts a fine control of vascular smooth muscle tone and arterial blood pressure, and plays a significant cardioprotective effect. This has been lately confirmed in experimental studies employing transgenic mice overexpressing human tissue kallikrein and animals with knockout of kinin B(1) and B(2) receptor gene. Disturbances in this system are associated with arterial hypertension, myocardial ischaemia and other clinical complications. Inhibitors of kininase II (angiotensin-converting enzyme) have been prescribed successfully to patients with cardiovascular diseases, but there is still a great interest in developing drugs or pharmacological strategies that augment the activity of kininogen-kallikrein-kinin system in pathological conditions. Delivery of adenovirus vector containing the human tissue kallikrein gene (gene kallikrein therapy) has emerged as a great potential to satisfy these conditions. This review provides a summary of plasma and tissue kallikrein-kinin system, focusing on the pharmacological properties, kinin receptors and drugs reported to interfere with their actions. The modulatory effects of the kallikrein-kinin system on cardiovascular system, particularly in regulating smooth muscle tone and arterial blood pressure and in preventing myocardium ischaemia have also been explored in the review.
Assuntos
Bradicinina/fisiologia , Sistema Cardiovascular/metabolismo , Sistema Calicreína-Cinina/fisiologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Bradicinina/biossíntese , Bradicinina/metabolismo , Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/efeitos dos fármacos , Terapia Genética , Humanos , Calidina/biossíntese , Calidina/metabolismo , Sistema Calicreína-Cinina/efeitos dos fármacos , Cininogênios/metabolismo , Cininas/antagonistas & inibidores , Cininas/genética , Cininas/metabolismo , Calicreína Plasmática/antagonistas & inibidores , Calicreína Plasmática/metabolismo , Inibidores de Proteases/uso terapêutico , Calicreínas Teciduais/antagonistas & inibidores , Calicreínas Teciduais/metabolismoRESUMO
Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.
Assuntos
Caesalpinia/química , Fator XIIa/antagonistas & inibidores , Fibrinolisina/antagonistas & inibidores , Calicreína Plasmática/antagonistas & inibidores , Sementes/química , Inibidores de Serina Proteinase/isolamento & purificação , Sequência de Aminoácidos , Caesalpinia/embriologia , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologiaRESUMO
Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).
Assuntos
Fator XII/efeitos dos fármacos , Fibrinolíticos/farmacologia , Glicosaminoglicanos/farmacologia , Calicreína Plasmática/efeitos dos fármacos , Plasminogênio/efeitos dos fármacos , Animais , Bovinos , Proteínas Inativadoras do Complemento 1/efeitos dos fármacos , Proteína Inibidora do Complemento C1 , Inibidores de Cisteína Proteinase/farmacologia , Fator XII/fisiologia , Humanos , Calicreína Plasmática/antagonistas & inibidores , Calicreína Plasmática/fisiologiaRESUMO
A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.