Peptides et pseudopeptides auto-assembleurs à activité antimicrobienne

La résistance des pathogènes contre les antibiotiques conventionnels augmente à un taux qui excède de loin l'allure du développement de nouveaux médicaments. Les peptides antimicrobiens, tant synthétiques que provenant de sources naturelles, se profilent comme étant des candidats avec lesquels il faudra compter. L utilisation des peptides est cependant limitée par une élimination rapide en milieu biologique liée à leur dégradation par les protéases. L'objet de cette thèse concerne la synthèse de monomères aza-b3-aminoacides dans l'optique de les incorporer dans des séquences peptidiques antimicrobiennes. Les aza-b3-aminoacides sont des analogues des a-aminoacides qui diffèrent des b3-aminoacides par la présence d'un atome d'azote supplémentaire chiral à configuration non fixée qui porte la chaîne latérale. L'introduction de ces unités monomériques au sein de peptides antimicrobiens donne accès à des pseudopeptides antimicrobiens dont la durée de vie et l'activité est améliorée. La modification d'un neuropeptide de seiche présentant des activités antibactériennes nous a permis d'obtenir un analogue peptidique et deux analogues pseudopeptidiques plus actifs que le peptide naturel. D'un autre côté, nous avons développé un peptide et une série de cyclopseudopeptides hybrides de novo qui possèdent des activités comparables à certains antibiotiques usuels. Nous avons tenté d'expliquer le mécanisme d'action de nos peptides et pseudopeptides qui agissent sur les membranes microbiennes. Les résultats obtenus par dichroïsme circulaire, diffusion de la lumière et RMN mettent en évidence un phénomène d'auto-association qui jouerait un rôle primordial dans l'activité biologique.Antibiotic resistance of pathogens against conventional antibiotics increases at a rate which exceeds by far the speed of the development of new drugs. The antimicrobial peptides, both synthetic and from natural sources, have raised interest as potential useful drugs in the future. The use of peptides is however limited by a fast elimination in biological environment connected to their rapid degradation by proteases. The object of this work concerns the synthesis of no proteinogenic monomers, the aza-b3-aminoacids, in the goal to incorporate them into antimicrobial peptidic sequences. Aza-b3-aminoacid are analogues of a-aminoacids, which differ from b3-aminoacids by the presence of an extra nitrogen atom, with a non-fixed configuration, which carries the side chain. The introduction of these monomeric units within antimicrobial peptides gives access to antimicrobial pseudopeptides with an extended life cycle. The modification of a cuttlefish neuropeptide presenting antibacterial activities allowed us to obtain a peptidic analogue and two pseudopeptidic analogues more active than the natural peptide. On the other hand, we developed a peptide and a series of cyclopseudopeptides hybrids de novo which possess comparable activities to some common antibiotics. Now, we try to explain the mechanism of action of our peptides and pseudopeptides which act on the microbial membranes. The results, obtained by circular dichroism, light scattering and NMR, bring to light a phenomenon of auto-association which would play an essential role in the biological activity.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Allosteric monofunctional aspartate kinases from Arabidopsis.

International audiencePlant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form

De Novo Cyclic Pseudopeptides Containing Aza-β3-amino Acids Exhibiting Antimicrobial Activities.

International audienceDe novo cyclic pseudopeptides composed of α-amino and aza-β3-amino acids were designed with the aim to obtain potential new antimicrobial agents. Antimicrobial cyclic pseudopeptides (ACPPs) are based on the properties of antimicrobial peptides (AMPs), so they are cationic and amphiphilic. Aza-β3-amino acids enhance the in vivo half-life of these compds. and offer the possibility to incorporate a large variety of side chains. Most of the 13 ACPPs exert antimicrobial activities in rich media with broad spectrum of antibacterial activities. Selectivity for bacterial over mammalian cells was detd. by testing the hemolytic activities of ACPPs against sheep red blood cells (sRBC). We examd. the ratio of cationic to hydrophobic residues as well as the type of hydrophobic side chains essential for biol. activity of this class of ACPPs. These results will be useful for designing potential candidates for a therapeutic application. [on SciFinder(R)

KKKKPLFGLFFGLF: A cationic peptide designed to exert antibacterial activity

International audienceWith 14 residues organized as two domains linked by a single proline, the de novo peptide called K4 was designed, using Antimicrobial Peptide Database, to exert antibacterial activity. The N-terminal domain is composed of four lysines enhancing membrane interactions, and the C-terminal domain is putatively folded into a hydrophobic alpha-helix. Following the synthesis, the purification and the structural checking, antibacterial assays revealed a strong activity against gram-positive and gram-negative bacteria including human pathogenic bacteria such as Staphylococcus aureus and some marine bacteria of the genus Vibrio. Scanning electron microscopy of Escherichia coli confirmed that K4 lyses bacterial cells. The cytotoxicity was tested against rabbit erythrocytes and chinese hamster ovary cells (CHO-K1). These tests revealed that K4 is non-toxic to mammalian cells for bacteriolytic concentrations. The peptide K4 could be a valuable candidate for future therapeutic applications

Enhancement of Antimicrobial Activity of a Natural Antimicrobial Neuropeptide of Sepia officinalis.

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