RESUMO
Antimicrobial peptides are valuable agents to fight antibiotic resistance. These amphipatic species display positively charged and hydrophobic amino acids. Here, we enhance the local hydrophobicity of a model peptide derived from human lysozyme (107RKWVWWRNR115) by arylation of its tryptophan (Trp) residues, which renders a positive effect on Staphylococcus aureus and Staphylococcus epidermidis growth inhibition. This site-selective modification was accessed by solid-phase peptide synthesis using the non-proteinogenic amino acid 2-aryltryptophan, generated by direct C-H activation from protected Trp. The modification brought about a relevant increase in growth inhibition: S. aureus was fully inhibited by arylation of Trp 112 and by only 10% by arylation of Trp 109 or 111, respect to the non-arylated peptide. On the other hand, S. epidermidis was fully inhibited by the three arylated peptides and the parent peptide. The minimum inhibitory concentration was significantly reduced for S. aureus depending on the arylation site.
Assuntos
Antibacterianos/farmacologia , Muramidase/química , Fragmentos de Peptídeos/farmacologia , Triptofano/química , Antibacterianos/química , Humanos , Testes de Sensibilidade Microbiana , Muramidase/farmacologia , Fragmentos de Peptídeos/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Desmopressin (dDAVP) is a safe haemostatic agent with previously reported antitumour activity. It acts as a selective agonist for the V2 vasopressin membrane receptor (V2r) present on tumour cells and microvasculature. The purpose of this study was to evaluate the novel peptide derivative [V4Q5]dDAVP in V2r-expressing preclinical mouse models of breast cancer. We assessed antitumour effects of [V4Q5]dDAVP using human MCF-7 and MDA-MB-231 breast carcinoma cells, as well as the highly metastatic mouse F3II cell line. Effect on in vitro cancer cell growth was evaluated by cell proliferation and clonogenic assays. Cell cycle distribution was analysed by flow cytometry. In order to study the effect of intravenously administered [V4Q5]dDAVP on tumour growth and angiogenesis, breast cancer xenografts were generated in athymic mice. F3II cells were injected into syngeneic mice to evaluate the effect of [V4Q5]dDAVP on spontaneous and experimental metastatic spread. In vitro cytostatic effects of [V4Q5]dDAVP against breast cancer cells were greater than those of dDAVP, and associated with V2r-activated signal transduction and partial cell cycle arrest. In MDA-MB-231 xenografts, [V4Q5]dDAVP (0.3 µg/kg, thrice a week) reduced tumour growth and angiogenesis. Treatment of F3II mammary tumour-bearing immunocompetent mice resulted in complete inhibition of metastatic progression. [V4Q5]dDAVP also displayed greater antimetastatic efficacy than dDAVP on experimental lung colonisation by F3II cells. The novel analogue was well tolerated in preliminary acute toxicology studies, at doses ≥ 300-fold above that required for anti-angiogenic/antimetastatic effects. Our data establish the preclinical activity of [V4Q5]dDAVP in aggressive breast cancer, providing the rationale for further clinical trials.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Desamino Arginina Vasopressina/análogos & derivados , Receptores de Vasopressinas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desamino Arginina Vasopressina/administração & dosagem , Desamino Arginina Vasopressina/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The synthetic nonapeptide 1desamino8Darginine vasopressin (dDAVP) can reduce tumor cell growth through agonist action on the vasopressin V2 receptor. A structureantiproliferative activity relationship analysis of dDAVP was performed using the alanine scanning technique on the aggressive MDAMB231 human breast carcinoma cell line. The results from this analysis demonstrated that the amino acids located at the loop of dDAVP are important for the antiproliferative activity of dDAVP, highlighting the key role of the Nterminal region of the peptide in the interaction with the tumor cell surface receptor. The findings from this study present novel strategies for designing improved compounds with enhanced stability for cancer therapy.
Assuntos
Desamino Arginina Vasopressina/química , Receptores de Vasopressinas/química , Relação Estrutura-Atividade , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desamino Arginina Vasopressina/análogos & derivados , Desamino Arginina Vasopressina/farmacologia , Humanos , Receptores de Vasopressinas/agonistas , Ensaio Tumoral de Célula-TroncoRESUMO
Substitution of Ala 108 and Ala 111 in the 107-115 human lysozyme (hLz) fragment results in a 20-fold increased anti-staphylococcal activity while its hemolytic activity becomes significant (30%) at very high concentrations. This analog displays an additional positive charge near the N-terminus (108) and an extra Trp residue at the center of the molecule (111), indicating that this particular amino acid sequence improves its interaction with the bacterial plasma membrane. In order to understand the role of this arrangement in the membrane interaction, studies with model lipid membranes were carried out. The interactions of peptides, 107-115 hLz and the novel analog ([K(108)W(111)]107-115 hLz) with liposomes and lipid monolayers were evaluated by monitoring the changes in the fluorescence of the Trp residues and the variation of the monolayers surface pressure, respectively. Results obtained with both techniques revealed a significant affinity increase of [K(108)W(111)]107-115 hLz for lipids, especially when the membranes containing negatively charged lipids, such as phosphatidylglycerol. However, there is also a significant interaction with zwitterionic lipids, suggesting that other forces in addition to electrostatic interactions are involved in the binding. The analysis of adsorption isotherms and the insertion kinetics suggest that relaxation processes of the membrane structure are involved in the insertion process of novel peptide [K(108)W(111)]107-115 hLz but not in 107-115 hLz, probably by imposing a reorganization of water at the interphases. In this regard, the enhanced activity of peptide [K(108)W(111)]107-115 hLz may be explained by a synergistic effect between the increased electrostatic forces as well as the increased hydrophobic interactions.
Assuntos
Antibacterianos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Eletricidade Estática , Sequência de Aminoácidos , Fluorescência , Humanos , Cinética , Bicamadas Lipídicas/química , Membranas Artificiais , Dados de Sequência Molecular , Fosfolipídeos/química , Pressão , Alinhamento de Sequência , Triptofano/metabolismoRESUMO
The most challenging target in the design of new antimicrobial agents is the development of antibiotic resistance. Antimicrobial peptides are good candidates as lead compounds for the development of novel anti-infective drugs. Here we propose the sequential substitution of each Ala residue present in a lead peptide with known antimicrobial activity by specific amino acids, rationally chosen, that could enhance the activity of the resultant peptide. Taking the fragment 107-115 of the human lysozyme as lead, two-round screening by sequentially replacing both Ala residues (108 and 111) by distinct amino acids resulted in a novel peptide with 4- and 20-fold increased antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. These results reinforce the strategy proposed, which, in combination with simple and easy screening tools, will contribute to the rapid development of new therapeutic peptides required by the market.
Assuntos
Alanina/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Muramidase/química , Muramidase/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Anti-Infecciosos/síntese química , Escherichia coli/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25 degrees C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8 degrees C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25 degrees C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.