RESUMO
Despite the need for innovative compounds as antimicrobial and anticancer agents, natural sources of peptides remain underexplored. Protonectin (PTN), a cationic dodecapeptide of pharmacological interest, presents large hydrophobicity that is associated with the tendency to aggregate and supposedly influences bioactivity. A disaggregating role was assigned to PTN' N-terminal fragment (PTN1-6), which enhances the bioactivity of PTN in a 1:1 mixture (PTN/PTN1-6). Spectroscopic techniques and model membranes (phospholipid bilayers and SDS micelles) revealed that environment-dependent aggregation is reduced for PTN/PTN1-6, but cytotoxicity of PTNs on MDA-MB-231 breast cancer showed the same CC50 values around 16 µM and on MCF-10A epithelial breast cells 6 to 5-fold higher values, revealing a selective interaction. Since PTN1-6 lacks activity on breast cells, its presence should differently affect PTN activity, suggesting that aggregation could modulate activity depending on the membrane characteristics. Indeed, increased partitioning and lytic activity of PTN/PTN1-6 were found in model membranes independently of charge density, but affected by the curvature tendency. PTN and PTN/PTN1-6 do not alter morphology and roughness of cancer cells, indicating a superficial interaction with membranes and consistent with results obtained in NMR experiments. Our results indicate that aggregation of PTNs depends on the membrane characteristics and modulates the activity of the peptides.
Assuntos
Antineoplásicos , Neoplasias da Mama , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Lipídeos , MicelasRESUMO
Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches from minimalist sequences and endogenous peptides modifications to computational libraries. Evaluation relies on microbiological tests. Aiming a deeper understanding, we chose the octapeptide Jelleine-I (JI) for its selective and low toxicity profile, designed small modifications combining the substitutions of Phe by Trp and Lys/His by Arg and tested the antimicrobial and anticancer activity on melanoma cells. Biophysical methods identified environment-dependent modulation of aggregation, but critical aggregation concentrations of JI and analogs in buffer show that peptides start membrane interactions as monomers. The presence of model membranes increases or reduces the partial aggregation of peptides. Compared to JI, analog JIF2WR shows the lowest tendency to aggregation on bacterial model membranes. JI and analogs are lytic to model membranes. Their composition-dependent performance indicates preference for the higher charged anionic bilayers in line with their superior performance toward Staphylococcus aureus and Streptococcus pneumoniae. JIF2WR presented the higher partitioning, higher lytic activity and lower aggregated contents. Despite these increased membranolytic activities, JIF2WR exhibited comparable antimicrobial activity in relation to JI at the expenses of some loss in selectivity. We found that the substitution Phe/Trp (JIF2W) tends to decrease antimicrobial but to increase anticancer activity and aggregation on model membranes and the toxicity toward human cells. However, the concomitant substitution Lys/His by Arg (JIF2WR) modulates some of these tendencies, increasing both the antimicrobial and the anticancer activity while decreasing the aggregation tendency.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Antineoplásicos/farmacologia , Membrana Celular/metabolismo , Hemólise/efeitos dos fármacos , Melanoma/patologia , Oligopeptídeos/toxicidade , Animais , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Arginina/química , Candida/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Camundongos , Oligopeptídeos/química , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Triptofano/químicaRESUMO
Solitary wasps use their stinging venoms for paralyzing insect or spider prey and feeding them to their larvae. We have surveyed bioactive substances in solitary wasp venoms, and found antimicrobial peptides together with some other bioactive peptides. Eumenine mastoparan-AF (EMP-AF) was the first to be found from the venom of the solitary eumenine wasp Anterhynchium flavomarginatum micado, showing antimicrobial, histamine-releasing, and hemolytic activities, and adopting an α-helical secondary structure under appropriate conditions. Further survey of solitary wasp venom components revealed that eumenine wasp venoms contained such antimicrobial α-helical peptides as the major peptide component. This review summarizes the results obtained from the studies of these peptides in solitary wasp venoms and some analogs from the viewpoint of (1) chemical and biological characterization; (2) physicochemical properties and secondary structure; and (3) channel-like pore-forming properties.
Assuntos
Anti-Infecciosos/química , Conformação Proteica em alfa-Hélice , Venenos de Vespas/análise , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Venenos de Vespas/química , Venenos de Vespas/farmacologiaRESUMO
BACKGROUND: Leishmaniasis threatens poor areas population worldwide, requiring new drugs less prone to resistance development. Antimicrobial peptides with antileishmanial activity are considered among fulfilling alternatives, but not much is known about the mode of action of membrane-targeting peptides, considering promastigote and infected macrophage membranes. In a previous work, structural features of very active known peptides were prospected using molecular dynamics simulations. METHODS: Combining sequences of these peptides, analogs were designed. The structure of analog DecP-11 was validated by NMR. In vitro bioassays determined the peptide cytotoxicity toward mammalian cells, IC50 values on promastigotes and amastigotes, and membranolytic activity compared to Decoralin, one of the parent peptides. With biophysical methods, the mechanism of interaction with membrane mimetic systems was investigated. RESULTS: The designed peptide exhibits potent cytolytic and membrane permeabilizing activities, and decreased antileishmanial activity compared to the parent peptide. Interactions with lipid bilayers mimicking those of promastigotes, infected macrophage and mammalian cells showed that these peptides strongly bind to vesicles with intense lytic activity at low concentrations. Additionally, circular dichroism and light scattering experiments showed changes in the secondary structure of peptides and in vesicle size, depending on vesicles compositions. Altogether they suggest that DecP-11 antileishmanial activity is impaired by the aggregation and that aminophospholipids are probably involved. CONCLUSIONS: DecP-11 potent cytolytic and membranolytic activities with lack of selectivity toward promastigote model membranes warrant further structural studies to improve selectivity. GENERAL SIGNIFICANCE: Strong interactions of peptides with aminophospholipids, abundant in parasite membranes, potentially lead to aggregated forms impairing activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Biofísica , Membrana Celular/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Sequência de Aminoácidos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Membrana Celular/química , Dicroísmo Circular , Leishmaniose/parasitologia , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Leishmaniasis, a protozoan-caused disease, requires alternative treatments with minimized side-effects and less prone to resistance development. Antimicrobial peptides represent a possible choice to be developed. We report on the prospection of structural parameters of 23 helical antimicrobial and leishmanicidal peptides as a tool for modeling and predicting the activity of new peptides. This investigation is based on molecular dynamic simulations (MD) in mimetic membrane environment, as most of these peptides share the feature of interacting with phospholipid bilayers. To overcome the lack of experimental data on peptides' structures, we started simulations from designed 100% α-helices. This procedure was validated through comparisons with NMR data and the determination of the structure of Decoralin-amide. From physicochemical features and MD results, descriptors were raised and statistically related to the minimum inhibitory concentration against Leishmania by the multivariate data analysis technique. This statistical procedure confirmed five descriptors combined by different loadings in five principal components. The leishmanicidal activity depends on peptides' charge, backbone solvation, volume, and solvent-accessible surface area. The generated model possesses good predictability (q2 = 0.715, r2 = 0.898) and is indicative for the most and the least active peptides. This is a novel theoretical path for structure-activity studies combining computational methods that identify and prioritize the promising peptide candidates.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Desenho de Fármacos , Humanos , Leishmaniose/tratamento farmacológico , Simulação de Dinâmica Molecular , Análise MultivariadaRESUMO
Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function.
Assuntos
Ácidos Araquidônicos/farmacologia , Membrana Celular/metabolismo , Endocanabinoides/farmacologia , Proteínas de Membrana/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Membrana Celular/efeitos dos fármacos , Gramicidina/farmacologia , Bicamadas Lipídicas/química , Fosfatidilcolinas/químicaRESUMO
Protonectin (ILGTILGLLKGL-NH2), a peptide extracted from the venom of the wasp Agelaia pallipes pallipes, promotes mast cell degranulation activity, antibiosis against Gram-positive and -negative bacteria, and chemotaxis in polymorphonucleated leukocytes. Another peptide from the same venom, Protonectin (1-6), corresponding to the first six residues of Protonectin, exhibits only chemotaxis. A 1:1 mixture of these two peptides showed positive synergistic antimicrobial effects, attributed to the formation of a heterodimer.16 The antimicrobial activity is probably related to the peptides' interaction with membrane phospholipids. Equilibrium and replica exchange molecular dynamics simulations were used to investigate two systems: the interaction of Protonectins (two molecules) and that of a mixture Protonectin and Protonectin (1-6) in the environment of sodium dodecyl sulfate (SDS) micelles, which mimic bacterial membranes and are also highly anionic. We found that in both systems the peptides tend to aggregate in the aqueous environment and are held together by hydrophobic interactions and hydrogen bonds. In the equilibrium simulations, aggregated Protonectin/Protonectin (1-6) dissociates after penetrating the SDS micelle, whereas the two Protonectins remain associated throughout the simulation time. Also, in the replica exchange simulations, the Protonectins remain closer, associating through a greater number of hydrogen bonds, and were found at only one free energy minimum, whereas the peptides in the mixture display other probable distances from each other, which are significantly longer than those observed with two Protonectin molecules. Coulomb contributions and the free energy of the systems containing micelles were calculated and show that the interactions of the mixed peptides are favored, whereas the interactions between pure Protonectins are more probable. As a consequence of the preferential interaction with the micelle, the Protonectin molecule of the mixed system presents a higher helical structure content. The enhancement of the amphipathic features caused by Protonectin (1-6) can be related to the increase in the antimicrobial activity experimentally observed.
Assuntos
Oligopeptídeos/química , Venenos de Vespas/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Micelas , Modelos Moleculares , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Dodecilsulfato de Sódio/química , Relação Estrutura-Atividade , Venenos de Vespas/metabolismo , Venenos de Vespas/farmacologiaRESUMO
BACKGROUND: The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised - with an intra-molecular disulphide bridge; and reduced - in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now. METHODS: Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge. RESULTS: Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway. CONCLUSION: The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine. GENERAL SIGNIFICANCE: The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.
Assuntos
Antibacterianos/farmacologia , Quimiotaxia/efeitos dos fármacos , Edema/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Fragmentos de Peptídeos/química , Venenos de Vespas/farmacologia , Animais , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Células Cultivadas , Dicroísmo Circular , Edema/metabolismo , Hemólise/efeitos dos fármacos , Hiperalgesia/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxirredução , Fragmentos de Peptídeos/farmacologia , Ratos , Receptores de Leucotrienos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Vespas/química , Vespas/crescimento & desenvolvimentoRESUMO
This study shows that MP-1, a peptide from the venom of the Polybia paulista wasp, is more toxic to human leukemic T-lymphocytes than to human primary lymphocytes. By using model membranes and electrophysiology measurements to investigate the molecular mechanisms underlying this selective action, the porelike activity of MP-1 was identified with several bilayer compositions. The highest average conductance was found in bilayers formed by phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylserine (70:30). The presence of cholesterol or cardiolipin substantially decreases the MP-1 pore activity, suggesting that the membrane fluidity influences the mechanism of selective toxicity. The determination of partition coefficients from the anisotropy of Trp indicated higher coefficients for the anionic bilayers. The partition coefficients were found to be 1 order of magnitude smaller when the bilayers contain cholesterol or a mixture of cholesterol and sphingomyelin. The blue shift fluorescence, anisotropy values, and Stern-Volmer constants are indications of a deeper penetration of MP-1 into anionic bilayers than into zwitterionic bilayers. Our results indicate that MP-1 prefers to target leukemic cell membranes, and its toxicity is probably related to the induction of necrosis and not to DNA fragmentation. This mode of action can be interpreted considering a number of bilayer properties like fluidity, lipid charge, and domain formation. Cholesterol-containing bilayers are less fluid and less charged and have a tendency to form domains. In comparison to healthy cells, leukemic T-lymphocyte membranes are deprived of this lipid, resulting in decreased peptide binding and lower conductance. We showed that the higher content of anionic lipids increases the level of binding of the peptide to bilayers. Additionally, the absence of cholesterol resulted in enhanced pore activity. These findings may drive the selective toxicity of MP-1 to Jurkat cells.
Assuntos
Membrana Celular/efeitos dos fármacos , Leucemia/patologia , Bicamadas Lipídicas/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Linfócitos T/metabolismo , Venenos de Vespas/metabolismo , Venenos de Vespas/farmacologia , Vespas/química , Adsorção , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Membrana Celular/química , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Células Jurkat , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Porosidade , Ligação Proteica , Especificidade por Substrato , Propriedades de Superfície , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Venenos de Vespas/químicaRESUMO
In order to investigate the effect of the different positions of the positive charges generated by the ionization of the side-chain of lysine residues, on the structure-activity relationship of the mastoparans, the peptides Protonectarina-MP (INWKALLDAAKKVL-NH2), Parapolybia-MP (INWKKMAATALKMI-NH2) and Asn-2-Polybia-MP I (INWKKLLDAAKQIL-NH2) and MK-578 (INWLKAKKVAGMIL-NH2) were investigated as models. Thus, the four peptides had their secondary structure studied and were submitted to assays of mast cell degranulation, hemolysis, and antibiosis. The results of the bioassays made clear that those peptides bearing the positive charges positioned at the positions 4/5 and/or from 11 to 13 are the most active ones; meanwhile, the localization of the positive charges in the middle of peptide chain resulted in a poorly active peptide. Thus, Protonectarina-MP, Parapolybia-MP, and Asn-2-Polybia-MP I presented physiologically important hemolysis and antibiosis, while MK-578 presented only a reduced antibiotic activity. Circular dichroism analysis were carried-out in different environments revealing that the anionic environment of a mixture of phosphatidylcholine and phosphatidylglycerol (70:30) liposomes favored the higher helical content of the four peptides in this study in relation to the zwiterionic environment of 100% phosphatidylcholine liposomes. The positioning of the lysine residues at the strategic positions (4/5 and 11-13), flanking and maintaining stable α-helix which extends from the 4th to the 13th residue along the peptide chain, seems to contribute to maximal lytic efficiency of the mastoparans, which in turn results in a more homogeneous hydrophobic surface in the amphipathic structure.