Peptídeos antimicrobianos: síntese, ensaios biológicos, estudos termodinâmicos e análise estrutural por RMN em meios biomiméticos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

Many organisms employ noxious chemicals (e.g., amines, steroid derivatives, alkaloids and peptides) to defend themselves against predators and pathogenic microrganisms. Among these chemical agents stand out peptides that can be found in a wide variety of organisms including bacteria, fungi, plants, insects, fish, amphibians,arachnids and mammals. The vast majority of these peptides has antimicrobial property whose mechanism of action is independent of specific receptors and because of this, these molecules have been considered as an alternative to the available antibiotics. However, the mechanism through which peptides exert their antimicrobial activity is still not completely defined. Studies are needed to better understand the way these molecules act on bacterial cells. The present work describes the thermodynamic and structural studies of antimicrobial peptides in biomimetic environments to explain their mechanisms of interaction with bacterial membranes. In this study the peptides demadistictin K (DD K), homotarsinin (HMT-2), and LyeTx I were manually synthesized by solid phase procedure. DD K is an antimicrobial peptide which was previously isolated from skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to phosphatidylcholine liposomes was investigated by measurements of kinetics of carboxyfluorescein (CF) leakage, dynamic light scattering (DLS) and isothermal titration microcalorimetry (ITC). The rate and the extent of CF release were significantly reduced by the presence of cholesterol. DLS showed that the liposome size increases when titrated with DD K but addition of cholesterol reduces the liposome size increments. ITC studies also showed that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol which contributes to explain the low hemolytic activity of the peptide. The structural alterations of DD K were first investigated in the presence of membranes, micelles or in membrane-mimetic environments using circular dichroism (CD) spectroscopy. The DD K peptide clearly associates with membrane and adopts a high degree of helical conformation. Thus DDK conformation was studied in the presence of DPC micelles by bidimensional NMR spectroscopy in solution. These results indicate an amphipathic -helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. In this work it was also carried out structural, thermodynamic and biological studies of the antimicrobial peptide homotarsinin (HMT-2), isolated from frog species Phyllomedusa tarsius, and its monomeric chain (HMT-1). The natural peptide HMT-2 was more active against the bacteria Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa than the monomeric form HMT-1. The results indicated greater thermodynamic interaction of the dimeric peptide with POPC and POPG liposomes in relation to the monomeric peptide, corroborating the higher antimicrobial activity observed on HMT-2. The analysis of the CD results showed that HMT-2 has a higher propensity to adopt -helical conformation in comparison to HMT-1. Detailed studies of three-dimensional structures by NMR showed significant differences in the orientation of each monomeric chain of HMT-2 in aqueous and membrane environment. Whereas a more closed structure of HMT-2 in aqueous solution may indicate a protection mechanism of the peptide to proteases, a more opened structure is formed in DPC micelles, leading to greater surface contact between peptide and membrane. LyeTx I is a peptide isolated from the venom of Lycosa erythrognatha, also known as wolf spider, and in this work its antimicrobial activity and structural profile were investigated through CD and NMR techniques. Comparisons among LyeTx I and commercial drugs showed to be active against bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa). The CD results showed LyeTx I in presence of POPC containing cholesterol has a decrease in helical corformation content, and along with the observed low hemolytic activity in other studies, these results indicate that bacterial membranes seem to be the preferential targets rather than vertebrate membranes. The secondary structure of LyeTx I has shown a small randomcoil region at the N-terminus followed by an -helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction.

ASSUNTO(S)

produtos de ação antimicrobiana teses. peptidios sintese teses. química orgânica teses. sequencia de aminoacidos teses.

ACESSO AO ARTIGO

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