RESUMEN
Health is fundamental for the development of individuals and evolution of species. In that sense, for human societies is relevant to understand how the human body has developed molecular strategies to maintain health. In the present review, we summarize diverse evidence that support the role of peptides in this endeavor. Of particular interest to the present review are antimicrobial peptides (AMP) and cell-penetrating peptides (CPP). Different experimental evidence indicates that AMP/CPP are able to regulate autophagy, which in turn regulates the immune system response. AMP also assists in the establishment of the microbiota, which in turn is critical for different behavioral and health aspects of humans. Thus, AMP and CPP are multifunctional peptides that regulate two aspects of our bodies that are fundamental to our health: autophagy and microbiota. While it is now clear the multifunctional nature of these peptides, we are still in the early stages of the development of computational strategies aimed to assist experimentalists in identifying selective multifunctional AMP/CPP to control nonhealthy conditions. For instance, both AMP and CPP are computationally characterized as amphipatic and cationic, yet none of these features are relevant to differentiate these peptides from non-AMP or non-CPP. The present review aims to highlight current knowledge that may facilitate the development of AMP's design tools for preventing or treating illness.
Asunto(s)
Péptidos de Penetración Celular , Péptidos Antimicrobianos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/uso terapéutico , HumanosRESUMEN
Cell-penetrating peptides (CPPs) are naturally able to cross the lipid bilayer membrane that protects cells. These peptides share common structural and physicochemical properties and show different pharmaceutical applications, among which drug delivery is the most important. Due to their ability to cross the membranes by pulling high-molecular-weight polar molecules, they are termed Trojan horses. In this study, we proposed a machine learning (ML)-based framework named BChemRF-CPPred (beyond chemical rules-based framework for CPP prediction) that uses an artificial neural network, a support vector machine, and a Gaussian process classifier to differentiate CPPs from non-CPPs, using structure- and sequence-based descriptors extracted from PDB and FASTA formats. The performance of our algorithm was evaluated by tenfold cross-validation and compared with those of previously reported prediction tools using an independent dataset. The BChemRF-CPPred satisfactorily identified CPP-like structures using natural and synthetic modified peptide libraries and also obtained better performance than those of previously reported ML-based algorithms, reaching the independent test accuracy of 90.66% (AUC = 0.9365) for PDB, and an accuracy of 86.5% (AUC = 0.9216) for FASTA input. Moreover, our analyses of the CPP chemical space demonstrated that these peptides break some molecular rules related to the prediction of permeability of therapeutic molecules in cell membranes. This is the first comprehensive analysis to predict synthetic and natural CPP structures and to evaluate their chemical space using an ML-based framework. Our algorithm is freely available for academic use at http://comptools.linc.ufpa.br/BChemRF-CPPred .
Asunto(s)
Péptidos de Penetración Celular/química , Biología Computacional/métodos , Predicción/métodos , Algoritmos , Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos , Aprendizaje Automático , Modelos Teóricos , Redes Neurales de la Computación , Máquina de Vectores de SoporteRESUMEN
Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell types, translocate across plasma membranes and accumulate in the cell cytoplasm, organelles (e.g., the nucleus and mitochondria) and other subcellular compartments. CPPs are either of natural origin or de novo designed and synthesized from segments and patches of larger proteins or designed by algorithms. With such intrinsic properties, along with membrane permeation, translocation and cellular uptake properties, CPPs can intracellularly convey diverse substances and nanomaterials, such as hydrophilic organic compounds and drugs, macromolecules (nucleic acids and proteins), nanoparticles (nanocrystals and polyplexes), metals and radionuclides, which can be covalently attached via CPP N- and C-terminals or through preparation of CPP complexes. A cumulative number of studies on animal toxins, primarily isolated from the venom of arthropods and snakes, have revealed the cell-penetrating activities of venom peptides and toxins, which can be harnessed for application in biomedicine and pharmaceutical biotechnology. In this review, I aimed to collate examples of peptides from animal venoms and toxic secretions that possess the ability to penetrate diverse types of cells. These venom CPPs have been chemically or structurally modified to enhance cell selectivity, bioavailability and a range of target applications. Herein, examples are listed and discussed, including cysteine-stabilized and linear, α-helical peptides, with cationic and amphipathic character, from the venom of insects (e.g., melittin, anoplin, mastoparans), arachnids (latarcin, lycosin, chlorotoxin, maurocalcine/imperatoxin homologs and wasabi receptor toxin), fish (pardaxins), amphibian (bombesin) and snakes (crotamine and cathelicidins).
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Ponzoñas/metabolismo , Animales , Membrana Celular/metabolismo , Péptidos de Penetración Celular/aislamiento & purificación , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos , HumanosRESUMEN
Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/química , Albúmina Sérica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Sitios de Unión , Línea Celular , Péptidos de Penetración Celular/farmacología , Diabetes Mellitus Tipo 2/diagnóstico por imagen , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/diagnóstico por imagen , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , FN-kappa B/metabolismo , Células RAW 264.7 , Distribución Aleatoria , Distribución Tisular , Resultado del TratamientoRESUMEN
OBJECTIVE: To test cell penetrating and interfering peptide Mut3DPT-PP2A/SET in interaction between serine threonine phosphatase PP2A and its physiological inhibitor, the oncoprotein SET. MATERIALS AND METHODS: Adult male C3H/S-strain mice, 60 days old, were given a graft of breast adenocarcinoma cells (TN60) into subcutaneous tissue. Mut3DPT-PP2A/SET peptide was used to block PP2A and SET oncoprotein interaction. The graft-bearing animals were divided into a control group (injected with saline buffer), and an intervention group injected intraperitoneally with Mut3DPT-PP2A/SET peptide (5 mg/kg) every day from day 5 to day 37. The variables we used to compare the outcome in both groups were tumor size in mm (length×width) and histological changes. In the statistical analysis we used ANOVA and Student-Keuls multiple comparisons test and Tuckey for the post-test analysis. RESULTS: 48 mice were grafted at day 0 with breast UNLP-C3H/S tumor cells, and after randomization, they were assigned to one of the two study groups. At day 5 all mice were injected either with placebo or with the peptide. The treated group showed significant tumor reduction (p<0.07). Histological changes showed presence of apoptosis and necrosis of tumor in treated group. CONCLUSION: The peptide Mut3DPT-PP2A/SET has demonstrated anti-tumor activity by reduction in vivo of tumor growth becoming a promising future in anticancer therapy.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Péptidos de Penetración Celular/farmacología , Proteínas de Unión al ADN/efectos de los fármacos , Chaperonas de Histonas/efectos de los fármacos , Proteína Fosfatasa 2/efectos de los fármacos , Adenocarcinoma/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/metabolismo , Ratones , Necrosis , Péptidos/farmacología , Proteína Fosfatasa 2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Crotamine is a basic, 42-residue polypeptide from snake venom that has been shown to possess cell-penetrating properties. Here we describe the preparation, purification, biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine. We also describe the formation and characterization of crotamine-DNA and crotamine-RNA nanoparticles; and the delivery of these nanoparticles into cells and animals. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine-plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in living organisms such as mice, Plasmodium, and worms. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. We also describe here the design and characterization of crotamine-functionalized gold nanoparticles, and the delivery of these nanoparticles into cells. We also evaluated the viability of using the combination of crotamine with silica nanoparticles in animal models, aiming to provide slow delivery, and to decrease the crotamine doses needed for the biological effects. In addition, the efficacy of administering crotamine orally was also demonstrated.
Asunto(s)
Antineoplásicos/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Venenos de Crotálidos/administración & dosificación , Melanoma Experimental/tratamiento farmacológico , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Venenos de Crotálidos/química , Venenos de Crotálidos/farmacología , ADN/metabolismo , Colorantes Fluorescentes/química , Ratones , Nanopartículas , ARN/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CIGB-552 is a synthetic anti-tumor peptide capable of reducing tumor size and increasing the lifespan of tumor-bearing mice. Part of its anti-cancer effects consists of inducing apoptosis, modulating NF-kB signaling pathway, and the angiogenesis process. Although one of its major mediators, the COMMD1 protein, has been identified, the mechanism by which CIGB-552 exerts such effects remains elusive. In the present study, we show the role of COMMD1 in CIGB-552 mechanism of action by generating the COMMD1 knock-out from the human lung cancer cell line NCI-H460. A microarray was performed to analyze both wild-type and KO cell lines with regard to CIGB-552 treatment. Additionally, different signaling pathways were studied in both cell lines to validate the results. Furthermore, the interaction between CIGB-552 and COMMD1 was analyzed by confocal microscopy. By signaling pathway analysis we found that genes involved in cell proliferation and apoptosis, oncogenic transformation, angiogenesis and inflammatory response are potentially regulated by the treatment with CIGB-552. We then demonstrated that CIGB-552 is capable of modulating NF-kB in both 2D and 3D cell culture models. Finally, we show that the ability of CIGB-552 to negatively modulate NF-kB and HIF-1 pathways is impaired in the COMMD1 knock-out NCI-H460 cell line, confirming that COMMD1 is essential for the peptide mechanism of action.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Péptidos de Penetración Celular/farmacología , Neoplasias del Colon/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Neovascularización Patológica/inmunología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Células Tumorales CultivadasRESUMEN
INTRODUCTION: Controlled delivery of therapeutic molecules in a localized manner has become an area of interest due to its potential to reduce drug exposure to healthy tissues and consequently to minimize undesirable side effects. We have recently introduced novel cell-penetrating vehicles by immobilizing the antimicrobial peptide Buforin II (BUF-II) on magnetite nanoparticles (MPNPs). Despite the potent translocating abilities of such nanobioconjugates, they failed to preserve the antimicrobial activity of native BUF-II. In this work, we explored immobilization on MNPs with the aid of polymer surface spacers, which has been considered as an attractive alternative for the highly efficient conjugation of various biomolecules. METHODS: Here, we immobilized BUF-II on polyetheramine-modified magnetite nanoparticles to preserve its structural integrity. As a result, for the obtained nanobioconjugates the lost antimicrobial activity against gram-positive and gram-negative bacteria was only 50% with respect to the native BUF-II. The nanobioconjugates were also characterized via FTIR, DLS, TEM, and TGA. Delivery on THP-1, HaCaT, HFF, and Escherichia coli cells was conducted to confirm capability for cell membrane translocation. RESULTS: Colocalization with Lysotracker showed an endosomal escape efficiency of about 73∓12% in THP-1 cells. Avoidance of endocytic pathways of internalization was qualitatively confirmed by a delivery assay at low temperature. Nuclear penetration of the nanobioconjugates was corroborated via confocal microscopy and showed high biocompatibility as demonstrated by hemolysis levels below 5% and acute cytotoxicity of around 15%. CONCLUSION: The obtained nanobioconjugates were capable of translocating the cell membrane and nuclei of different normal and cancerous cell lines without significantly decreasing viability. This makes the vehicle addressable for a number of applications ranging from antimicrobial topical treatments to the delivery of nucleotides and therapeutic molecules with difficulties to bypass cell membranes.
Asunto(s)
Aminas/química , Antibacterianos/farmacología , Péptidos de Penetración Celular/farmacología , Nanopartículas de Magnetita/química , Nanoconjugados/química , Proteínas/farmacología , Antibacterianos/química , Materiales Biocompatibles/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/ultraestructura , Hemólisis/efectos de los fármacos , Humanos , Nanopartículas de Magnetita/ultraestructura , Proteínas/químicaRESUMEN
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.
Asunto(s)
Antiinfecciosos/química , Conformación Proteica en Hélice alfa , Venenos de Avispas/análisis , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Venenos de Avispas/química , Venenos de Avispas/farmacologíaRESUMEN
Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.
Asunto(s)
Péptidos de Penetración Celular , Venenos de Crotálidos , Células Epiteliales/metabolismo , Túbulos Renales Proximales/metabolismo , Animales , Péptidos de Penetración Celular/efectos adversos , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Venenos de Crotálidos/efectos adversos , Venenos de Crotálidos/farmacocinética , Venenos de Crotálidos/farmacología , Células Epiteliales/citología , Túbulos Renales Proximales/citología , Masculino , RatonesRESUMEN
Scorpion sting envenoming impacts millions of people worldwide, with cardiac effects being one of the main causes of death on victims. Here we describe the first Ca2+ channel toxin present in Tityus serrulatus (Ts) venom, a cell penetrating peptide (CPP) named CPP-Ts. We show that CPP-Ts increases intracellular Ca2+ release through the activation of nuclear InsP3R of cardiomyocytes, thereby causing an increase in the contraction frequency of these cells. Besides proposing a novel subfamily of Ca2+ active toxins, we investigated its potential use as a drug delivery system targeting cancer cell nucleus using CPP-Ts's nuclear-targeting property. To this end, we prepared a synthetic CPP-Ts sub peptide14-39 lacking pharmacological activity which was directed to the nucleus of specific cancer cell lines. This research identifies a novel subfamily of Ca2+ active toxins and provides new insights into biotechnological applications of animal venoms.
Asunto(s)
Calcio/química , Péptidos de Penetración Celular/química , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Secuencia de Aminoácidos/genética , Animales , Canales de Calcio , Línea Celular Tumoral , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/uso terapéutico , Citoplasma/efectos de los fármacos , Humanos , Venenos de Escorpión/química , Escorpiones/químicaRESUMEN
CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.
Asunto(s)
Antineoplásicos/farmacología , Péptidos de Penetración Celular/farmacología , Péptidos Cíclicos/farmacología , Péptidos/farmacología , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/síntesis química , Técnicas de Química Sintética/métodos , Humanos , Neoplasias/tratamiento farmacológico , Péptidos/síntesis química , Péptidos Cíclicos/síntesis química , Fosforilación/efectos de los fármacosRESUMEN
CIGB-552 is a twenty-amino-acid novel synthetic peptide that has proven to be effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Such capability is conferred by its cell-penetrating peptide character, which allows it to enter cells and elicit a pro-apoptotic effect through its major mediator, COMMD1 protein. Cell-penetrating peptides are able to use different internalization mechanisms, such as endocytosis or direct transduction through the plasma membrane. Although CIGB-552 cytotoxicity has been evaluated in several non-tumor- and tumor-derived cell lines, no data regarding the relationship between cell line sensitivity, cell penetrating capacity, the internalization mechanisms involved, COMMD1 expression levels, or its subcellular localization has yet been produced. Here, we present the results obtained from a comparative analysis of CIGB-552 sensitivity, internalization capacity and the mechanisms involved in three human tumor-derived cell lines from different origins: mammary gland, colon and lung (MCF-7, HT-29 and H460, respectively). Furthermore, cell surface markers relevant for internalization processes such as phosphatidylserine, as well as CIGB-552 target COMMD1 expression/localization, were also evaluated. We found that both endocytosis and transduction are involved in CIGB-552 internalization in the three cell lines evaluated. However, CIGB-552 incorporation efficiency and contribution of each mechanism is cell-line dependent. Finally, sensitivity was directly correlated with high internalization capacity in those cell lines where endocytosis had a major contribution on CIGB-552 internalization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos , Péptidos de Penetración Celular , Endocitosis/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Humanos , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Mature proteins can act as potential sources of encrypted bioactive peptides that, once released from their parent proteins, might interact with diverse biomolecular targets. In recent work we introduced a systematic methodology to uncover encrypted intragenic antimicrobial peptides (IAPs) within large protein sequence libraries. Given that such peptides may interact with membranes in different ways, resulting in distinct observable outcomes, it is desirable to develop a predictive methodology to categorize membrane active peptides and establish a link to their physicochemical properties. Building upon previous work, we explored the interaction of a range of IAPs with model membranes probed by differential scanning calorimetry (DSC) and circular dichroism (CD) techniques. The biophysical data were submitted to multivariate statistical methods and resulting peptide clusters were correlated to peptide structure and to their antimicrobial activity. A re-evaluation of the physicochemical properties of the peptides was conducted based on peptide cluster memberships. Our data indicate that membranolytic peptides produce characteristic thermal transition (DSC) profiles in model vesicles and that this can be used to categorize novel molecules with unknown biological activity. Incremental expansion of the model presented here might result in a unified experimental framework for the prediction of novel classes of membrane active peptides.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/clasificación , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Bacterias/metabolismo , Rastreo Diferencial de Calorimetría , Membrana Celular/química , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/clasificación , Péptidos de Penetración Celular/farmacología , Fenómenos Químicos , Humanos , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Conformación Proteica en Hélice alfaRESUMEN
Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.
Asunto(s)
Antifúngicos/química , Péptidos de Penetración Celular/química , Venenos de Crotálidos/química , Secuencia de Aminoácidos , Animales , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Candida/efectos de los fármacos , Candida/crecimiento & desarrollo , Péptidos de Penetración Celular/aislamiento & purificación , Péptidos de Penetración Celular/farmacología , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/farmacología , Crotalus/metabolismo , Cisteína/química , ADN/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Cinética , Pruebas de Sensibilidad Microbiana , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Unión Proteica , Electricidad Estática , Relación Estructura-Actividad , Trichosporon/efectos de los fármacos , Trichosporon/crecimiento & desarrollo , Liposomas Unilamelares/químicaRESUMEN
Linear cationic α-helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine-substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α-helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]10 -Dec-NH2 analog showed similar activity against different microorganisms (c.a. 0.4-0.8 µmol L-1 ), helical structuration, and some hemolytic activity, [Leu]8 -Dec-NH2 analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 µmol L-1 ). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Antibacterianos/farmacología , Péptidos de Penetración Celular/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antibacterianos/química , Bacillus subtilis/efectos de los fármacos , Candida albicans/efectos de los fármacos , Péptidos de Penetración Celular/química , Dicroismo Circular , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Hemólisis , Humanos , Leucina/química , Pruebas de Sensibilidad Microbiana , Micrococcus luteus/efectos de los fármacos , Conformación Proteica en Hélice alfa , Pseudomonas aeruginosa/efectos de los fármacos , Salmonella arizonae/efectos de los fármacosRESUMEN
PURPOSE: Regulatory T cells (Tregs) impair the clinical benefit of cancer immunotherapy. To optimize the antitumor efficacy of therapeutic dendritic cell (DC) vaccines, we aimed to inhibit Foxp3, a transcription factor required for Treg function. METHODS: Mice bearing established syngeneic LM3 and 4T1 breast tumors were treated with antitumor DC vaccines and a synthetic peptide (P60) that has been shown to inhibit Foxp3. RESULTS: Treatment with P60 improved the therapeutic efficacy of DC vaccines in these experimental models. In addition, monotherapy with P60 inhibited tumor growth in immunocompetent as well as in immuno-compromised animals bearing established tumors. We found expression of Foxp3 in human and murine breast tumor cells. P60 inhibited IL-10 secretion in breast cancer cells that expressed Foxp3. CONCLUSIONS: Our results suggest that Foxp3 blockade improves the therapeutic efficacy of DC vaccines by inhibition of Tregs and through a direct antitumor effect. This strategy could prove useful to neutralize the immunosuppressive microenvironment and to boost antitumor immunity in breast cancer.
Asunto(s)
Neoplasias de la Mama/terapia , Péptidos de Penetración Celular/administración & dosificación , Células Dendríticas/trasplante , Factores de Transcripción Forkhead/antagonistas & inhibidores , Linfocitos T Reguladores/efectos de los fármacos , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/farmacología , Línea Celular Tumoral , Péptidos de Penetración Celular/farmacología , Células Dendríticas/inmunología , Femenino , Humanos , Inmunoterapia , Ratones , Linfocitos T Reguladores/inmunología , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 µmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.
Asunto(s)
Antibacterianos/farmacología , Péptidos de Penetración Celular/farmacología , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli/efectos de los fármacos , Lactoferrina/farmacología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , HumanosRESUMEN
Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Aminoácidos/química , Antineoplásicos/farmacología , Péptidos de Penetración Celular/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Biotransformación , Caspasa 7/genética , Caspasa 7/metabolismo , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Expresión Génica , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Simulación de Dinámica Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.