RESUMO
The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus-MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3 kDa cut-off Amicon® ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73 Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.
Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Venenos de Crotalídeos/química , Peptídeos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antimaláricos/química , Antimaláricos/farmacologia , Bothrops , Venenos de Crotalídeos/isolamento & purificação , Hemolíticos/química , Hemolíticos/farmacologia , Humanos , Leishmania/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/isolamento & purificação , Plasmodium falciparum/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Staphylococcus aureus/efeitos dos fármacosRESUMO
The gold standard drug for colorectal cancer (CRC) treatment, 5-Fluorouracil (5-FU), induces pharmacological tolerance in long-term management. The transcriptional factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) plays a key role in 5-FU resistance. The aim of this work is to study the capability of polyelectrolytes complex nanoparticles of dermatan sulfate (DS) and chitosan (CS), loaded with the anti-inflammatory tripeptide IRW, to sensitize colorectal cancer cells to 5-FU. Fluorescence and flow cytometry studies confirmed the recognition by the nanoformulation, of the cluster of differentiation 44 (CD44) receptor, involved in the initiation and progression of colorectal tumors. Dynamic light scattering (DLS) and flow cytometry reinforced the importance of DS and CD44 receptor in the interaction, as the addition of DS or anti-CD44 antibody blocked the binding. Moreover, the nanoformulation also interacts with 3D colon cancer cultures, namely colonospheres, enriched in cancer stem cells (CSC), subpopulation responsible for drug resistance and metastasis. To evaluate the consequences of this interaction, the subcellular distribution of the transcriptional factor NFκB, is determined by immunofluorescence analysis. Internalization and the intracellular release of IRW inhibited nuclear translocation of NFκB and increased cellular sensitivity to 5-FU. Altogether, the nanoformulation could provide a selective delivery platform for IRW distribution to colorectal tumors, being an innovative strategy toward overcoming 5-FU resistance in CRC therapy.
Assuntos
Quitosana , Neoplasias Colorretais , Nanopartículas , Humanos , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Quitosana/farmacologia , Quitosana/uso terapêutico , Dermatan Sulfato/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , NF-kappa B , Peptídeos/uso terapêutico , Anti-Inflamatórios , Linhagem Celular TumoralRESUMO
Antimicrobial peptides (AMPs), also known as host defense peptides, are small and mostly polycationic molecules that form part of the innate immune response. There are currently more than 3000 experimentally reported AMPs. Particularly in frogs, the temporin family has been discovered as potential AMPs. The aim of this work is to review the latest publications about this class of peptides, discuss their properties, and present an update of the last studies and new discoveries in the field. More than 130 temporins have been identified in this family. The most studied temporins are temporin A (TA), temporin B (TB), and temporin L (TL). These peptides showed antimicrobial activity against gram-negative, gram-positive bacteria and fungi. Since the discovery of temporins in 1996, several groups of researchers isolated different peptides from various species of frogs that were included as members of this family. Although antimicrobial activity of many temporins has not been analyzed yet, most of them showed antimicrobial and antifungal activities. A combination of nanotechnology and AMPs for temporins in different antimicrobial treatments could be a promising alternative for resistant pathogens. These studies demonstrate that, even with the advancement in scientific research on the composition and antimicrobial activity of temporins, further studies are necessary to wholly understand their components and mechanisms of action.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Antifúngicos/química , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Anuros , HumanosRESUMO
Nowadays, treatment with specific antivenins is considered the only cure for snakebites accidents. However, access to antivenom obstructs the successful implementation of the World Health Organization international guidelines. In the last few years, natural organic compounds, peptides, and proteins with the ability to inhibit snake toxins and obtained from different sources such as plant extracts and animal blood have been proposed as antivenoms. In this work, we will focus on the inhibitors of the main venom toxins, phospholipases A2 and metalloproteinases, and their application as novel antivenoms.
Assuntos
Antivenenos/farmacologia , Produtos Biológicos/farmacologia , Venenos de Serpentes/antagonistas & inibidores , Animais , Antivenenos/química , Antivenenos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , HumanosRESUMO
We describe a method to develop affinity chromatography matrices with short peptide ligands for protein purification. The method entitles the following: (a) synthesis of a combinatorial library on the hydromethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry, (b) library screening with the protein of interest labeled with a fluorescent dye or biotin, (c) identification of peptides contained on positive beads by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), (d) solid-phase peptide ligand synthesis and immobilization in chromatographic supports, and (e) evaluation of protein adsorption on peptide affinity matrices from the equilibrium isotherms and breakthrough curves.