RESUMO
Acquired drug resistance is the major cause for disease recurrence in cancer patients, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. To address this problem, we investigated cyclic membrane-active peptides as an alternative therapeutic modality to kill drug-tolerant and resistant melanoma cells to avoid acquired drug resistance. We selected two stable cyclic peptides (cTI and cGm), previously shown to have anti-melanoma properties, and compared them with dabrafenib, a drug used to treat cancer patients with the BRAF V600E mutation. The peptides act via a fast membrane-permeabilizing mechanism and kill metastatic melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Melanoma cells do not become resistant to long-term treatment with cTI, nor do they evolve their lipid membrane composition, as measured by lipidomic and proteomic studies. In vivo studies in mice demonstrated that the combination treatment of cTI and dabrafenib resulted in fewer metastases and improved overall survival. Such cyclic membrane-active peptides are thus well suited as templates to design new anticancer therapeutic strategies.
Assuntos
Antineoplásicos , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Imidazóis , Melanoma , Oximas , Peptídeos Cíclicos , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Animais , Melanoma/tratamento farmacológico , Melanoma/patologia , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Oximas/farmacologia , Oximas/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Camundongos , Feminino , Proteínas de Ligação a DNARESUMO
Kalata B1 (kB1), a naturally occurring cyclotide has been shown experimentally to bind lipid membranes that contain phosphatidylethanolamine (PE) phospholipids. Here, molecular dynamics simulations were used to explore its interaction with two phospholipids, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), and a heterogeneous membrane comprising POPC/POPE (90:10), to understand the basis for the selectivity of kB1 towards PE phospholipids. The simulations showed that in the presence of only 10 % POPE lipid, kB1 forms a stable binding complex with membrane bilayers. An ionic interaction between the E7 carboxylate group of kB1 and the ammonium group of PE headgroups consistently initiates binding of kB1 to the membrane. Additionally, stable noncovalent interactions such as hydrogen bonding (E7, T8, V10, G11, T13 and N15), cation-π (W23), and CH-π (W23) interactions between specific residues of kB1 and the lipid membrane play an important role in stabilizing the binding. These findings are consistent with a structure-activity relationship study on kB1 where lysine mutagenesis on the bioactive and hydrophobic faces of the peptide abolished membrane-dependent bioactivities. In summary, our simulations suggest the importance of residue E7 (in the bioactive face) in enabling kB1 to recognize and bind selectively to PE-containing phospholipids bilayers through ionic and hydrogen bonding interactions, and of W23 (in the hydrophobic face) for the association and insertion of kB1 into the lipid bilayer through cation-π and CH-π interactions. Overall, this work enhances our understanding of the molecular basis of the membrane binding and bioactivity of this prototypic cyclotide.
Assuntos
Ciclotídeos , Fosfolipídeos , Simulação de Dinâmica Molecular , Fosfatidiletanolaminas/química , Ciclotídeos/química , Ciclotídeos/metabolismo , CátionsRESUMO
Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Proteínas de Ligação a DNA/farmacologia , Peptídeos Cíclicos/farmacologia , Animais , Células da Medula Óssea , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos , Caranguejos Ferradura/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Cultura Primária de CélulasRESUMO
Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos , Neoplasias/metabolismo , Aranhas/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/farmacocinética , Proteínas de Artrópodes/farmacologia , Membrana Celular/patologia , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
Melioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current antibiotic regimens used to treat melioidosis are prolonged and expensive, and often ineffective because of intrinsic and acquired antimicrobial resistance. Efforts to develop new treatments for melioidosis are limited by the risks associated with handling pathogenic B. pseudomallei, which restricts research to facilities with biosafety level three containment. Closely related nonpathogenic Burkholderia can be investigated under less stringent biosafety level two containment, and we hypothesized that they could be used as model organisms for developing therapies that would also be effective against B. pseudomallei. We used microbroth dilution assays to compare drug susceptibility profiles of three B. pseudomallei strains and five nonpathogenic Burkholderia strains. Burkholderia humptydooensis, Burkholderia thailandensis, and Burkholderia territorii had similar susceptibility profiles to pathogenic B. pseudomallei that support their potential as safer in vitro models for developing new melioidosis therapies.
Assuntos
Antibacterianos/farmacologia , Burkholderia/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Melioidose/tratamento farmacológico , Burkholderia/genética , Burkholderia/patogenicidade , Humanos , Especificidade da EspécieRESUMO
Mimics of natural antimicrobial peptides are promising compounds to fight the rising threat of multi-drug resistant bacteria. Here we report the design, synthesis and conformational analysis of a new class of antimicrobial peptide mimetics incorporating a diphenylacetylene scaffold. Within a small set of compounds, we observe a correlation between amphiphilicity, the efficiency of partitioning into negatively charged membranes and antibacterial activity. The most amphiphilic compound, which contains four isoleucine residues and four lysine residues, displays species-selective antibacterial activity (most active against Bacillus subtills) and low haemolytic activity. Solution-phase conformational analysis of this compound indicates that a defined structure is adopted in the presence of negatively charged phospholipid membranes and aqueous 2,2,2-trifluoroethanol but not in water. A conformation model indicates that the cationic and hydrophobic functional groups are segregated. These results may inform the development of highly selective antimicrobial peptide mimetics for therapeutic applications.
Assuntos
Alcinos/farmacologia , Antibacterianos/farmacologia , Peptidomiméticos/farmacologia , Alcinos/síntese química , Animais , Antibacterianos/síntese química , Bactérias/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Bicamadas Lipídicas/química , Lipossomos/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Peptidomiméticos/síntese química , CoelhosRESUMO
Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.
Assuntos
Apoptose , Imageamento Tridimensional , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Fagocitose/efeitos dos fármacos , Fosfatidilserinas/metabolismoRESUMO
Cyclotides are plant-derived peptides characterized by an â¼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclotídeos/farmacologia , Descoberta de Drogas , Plantas Medicinais/química , Violaceae/química , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em TandemRESUMO
Antimicrobial peptides are an attractive alternative to traditional antibiotics, due to their physicochemical properties, activity toward a broad spectrum of bacteria, and mode-of-actions distinct from those used by current antibiotics. In general, antimicrobial peptides kill bacteria by either disrupting their membrane, or by entering inside bacterial cells to interact with intracellular components. Characterization of their mode-of-action is essential to improve their activity, avoid resistance in bacterial pathogens, and accelerate their use as therapeutics. Here we review experimental biophysical tools that can be employed with model membranes and bacterial cells to characterize the mode-of-action of antimicrobial peptides.
RESUMO
Peptides with pharmaceutical activities are attractive drug leads, and knowledge of their mode-of-action is essential for translation into the clinic. Comparison of native and enantiomeric peptides has long been used as a powerful approach to discriminate membrane- or receptor-mediated modes-of-action on the basis of the assumption that interactions with cell membranes are independent of peptide chirality. Here, we revisit this paradigm with the cyclotide kalata B1, a drug scaffold with intrinsic membrane-binding activity whose enantiomer is less potent than native peptide. To investigate this chirality dependence, we compared peptide-lipid binding using mirror image model membranes. We synthesized phospholipids with non-natural chirality and demonstrate that native kalata B1 binds with higher affinity to phospholipids with chirality found in eukaryotic membranes. This study shows for the first time that the chiral environment of lipid bilayers can modulate the function of membrane-active peptides and challenges the view that peptide-lipid interactions are achiral.
Assuntos
Membrana Celular/química , Ciclotídeos/química , Fosfolipídeos/química , Sítios de Ligação , Ciclotídeos/síntese química , Ciclotídeos/isolamento & purificação , Células Eucarióticas/citologia , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares/citologia , Modelos MolecularesRESUMO
Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (NaVs) involved in pain signal transmission, including the NaV1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of NaV that are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit NaV1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other NaV subtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described NaV1.7 potent/selective GpTx-1 analogues and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for NaV1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes and that altering these residues can have dramatic effects on NaV inhibitory activity. We demonstrate that strong peptide-membrane interactions are not essential for inhibiting NaV1.7 and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed NaV residues, thereby affecting peptide-channel interactions. Our detailed structure-activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for NaV1.7, as single mutations can induce local changes in GMT structure that can have a major impact on NaV-inhibitory activity.
Assuntos
Peptídeos/efeitos dos fármacos , Venenos de Aranha/farmacologia , Animais , Humanos , Peptídeos/químicaRESUMO
Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.
Assuntos
Venenos de Cnidários/química , Peptídeos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Venenos de Cnidários/genética , Venenos de Cnidários/metabolismo , Evolução Molecular , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Conformação Proteica , Dobramento de Proteína , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/metabolismo , TranscriptomaRESUMO
Malaria is a serious threat to human health and additional classes of antimalarial drugs are greatly needed. The human defense protein, platelet factor 4 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial domain, which through cyclization, retained the critical structure of the parent protein. The peptide, cPF4PD, killed cultured blood-stage Plasmodium falciparum with low micromolar potency by specific disruption of the parasite digestive vacuole. Its mechanism of action involved selective penetration and accumulation inside the intraerythrocytic parasite without damaging the host cell or parasite membranes; it did not accumulate in uninfected cells. This selective activity was accounted for by observations of the peptide's specific binding and penetration of membranes with exposed negatively charged phospholipid headgroups. Our findings highlight the tremendous potential of the cPF4PD scaffold for developing antimalarial peptide drugs with a distinct and selective mechanism of action.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Fator Plaquetário 4/química , Fator Plaquetário 4/farmacologia , Adulto , Desenho de Fármacos , Eritrócitos/parasitologia , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Modelos MolecularesRESUMO
Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Venenos de Aranha/farmacologia , Toxinas Biológicas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Aranhas/química , Toxinas Biológicas/química , Toxinas Biológicas/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Animais , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Humanos , Lipopeptídeos/efeitos adversos , Lipopeptídeos/uso terapêutico , Camundongos , Modelos Moleculares , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing â¼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.
Assuntos
Antibacterianos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular , Venenos de Crotalídeos , Crotalus , Escherichia coli , Fragmentos de Peptídeos , Pseudomonas aeruginosa , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Membrana Celular/química , Membrana Celular/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Escherichia coli/química , Escherichia coli/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.
Assuntos
Tensoativos/química , Sequência de Aminoácidos , Ciclotídeos , Bicamadas LipídicasAssuntos
Descoberta de Drogas/métodos , Peptídeos/química , Anticorpos/química , Anticorpos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Transporte Biológico , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Hormônios/química , Hormônios/uso terapêutico , Humanos , Biblioteca de Peptídeos , Peptídeos/uso terapêutico , Peptídeos Cíclicos/química , Peptídeos Cíclicos/uso terapêuticoRESUMO
Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus quinquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins.
Assuntos
Venenos de Escorpião/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Membrana Celular/química , Membrana Celular/metabolismo , Dissulfetos/química , Espectroscopia de Ressonância de Spin Eletrônica , Células HeLa , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lisina/química , Microscopia Confocal , Mutagênese , Estabilidade Proteica , Venenos de Escorpião/síntese química , Venenos de Escorpião/genética , Escorpiões/metabolismo , Alinhamento de SequênciaRESUMO
Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state 19F-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive 19F-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive 19F-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by 15N-NMR of uniformly labeled protein, and confirmed using solid-state 31P- and 2H-NMR. 31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2H-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the 19F-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.