Tesis doctoral de Anna Diaz Cirac
Antimicrobial peptides (amps) are currently in the spotlight as potential candidates to overcome bacterial resistance to conventional antibiotics. However, how these molecules kill bacteria by interacting with the cell membrane is not fully understood. Studying the biophysical processes of antimicrobial activity of these peptides can provide excellent information for the rational design of new improved candidates for the development of effective antibacterial agents. Amongst the large number of amps present in nature, cyclic peptides have emerged as good antimicrobial candidates due to their robust secondary structure and high activity. the starting point of this thesis is a library of de novo cyclic decapeptides which showed high antimicrobial activity against three plant pathogenic bacteria, namely pseudomonas syringae, erwinia amylovora and xanthomonas vesicatoria, and low hemolytic activity. These cyclic peptides consisted of alternating cationic (lysine) and hydrophobic (leucine and phenylalanine) amino acids, with a general formula of c(x5-phe-x3-gln) where x can be either lysine or leucine. in order to devise a general procedure for designing new cyclic antimicrobial candidates with improved activity, this work is devoted to the understanding of i) the factors governing the activity and ii) the mechanism of action at the atomistic level of these peptides, principally by making use of well-established computational molecular modeling tools such as molecular dynamics (md) simulations. this thesis is divided into two parts, one of them focused on the synthesis and evaluation of the biological activity of a series of cyclic amps, and another one where md simulations of these peptides were carried out in order to shed light on their conformational preferences, stability in water phase and their mechanism of action upon lipid membrane. in the first part, the experimental procedure covered the optimization of the synthesis of a particular cyclic decapeptide in different solid supports, namely resin and lanterns. We show how by changing the coupling reagents an improved head-to-tail cyclization over dimerization is reached. Also the influence of replacing the phenylalanine residue by a tryptophan for the cyclic peptides from the library is evaluated in detail. Even though the tryptophan analogue shows a higher antimicrobial activity, its cytotoxic activity is also affected giving rise to higher hemolytic activity. Moreover, the evaluation of the stability of some of the cyclic peptides in human serum is tested to study their potential therapeutic application for cancer therapy. Those active peptides prove to be more stable compared to their linear analogues. the second part of the thesis comprises the computational modeling of several specific cyclic amps from the library. In a first study, we describe a comprehensive conformational search of the cyclic peptide bpc16, c(klklkfklkq) by means of several strategies rooted on the use of md simulations. The analysis of the large set of conformers obtained revealed higher stability of structures with smaller radius of gyration. Dynamical studies showed a high stability of a compact structure both in gas phase and in solution. In the water phase, the effect of water molecules caused more fluctuations in the formation of hydrogen bonds but a stable secondary structure consisting of a beta-turn i and alpha-turn structure was established as the conformation of the peptide in solution. in a second study, and in collaboration with the molecular dynamics group of groningen, we used the best candidate from the library of cyclic peptides, bpc194 (c(kklkkfkklq)) and its non-active linear analogue (bpc193) to study the molecular basis for their antimicrobial activity. To do so, a number molecular dynamics simulations upon anionic lipid bilayer models were carried out. We show here that the cyclic peptide is able to fold in an amphypathic-like manner, which helps in the stabilization of a disordered toroidal pore. Furthermore, a structure-function relationship is derived from the careful analysis of a stable conformation of the peptides directly involved in the formation and stabilization of the porated state, best characterized by a $_x0008_eta$-structure with a spatially-symmetric arrangement of the lysine side-chains. In the last part of this section, bigger systems consisting of two sets of lipid bilayers were modeled in order to examine alternative modes of antimicrobial action. These simulations manifest the ability of the cyclic peptide bpc194 to both porate and fuse lipid membranes simultaneously. Such multi-mode process may be functionally relevant and contribute to the high effectiveness of this peptide towards bacteria killing. in the last part of this thesis we work with the hypothesis that the distinct structural motif found for the bpc194 peptide in the porated state plays a fundamental role in its antimicrobial activity, and therefore can be a key element for a rational design of new cyclic peptides with similar or enhanced activity. Principally based on the bioactive conformation of bpc194, nine cyclic peptides were designed and synthesized. Remarkably, peptide bpc490 exhibits improved activity against all three bacteria compared to the most active peptide of the original library, yet keeping its hemolytic activity still very low. such high antimicrobial activity found for some of these new peptides evidences the effectiveness of our rational design approach, by combining both experiment and computational modeling.
Datos académicos de la tesis doctoral «Mechanism of action of cyclic antimicrobial peptides«
- Título de la tesis: Mechanism of action of cyclic antimicrobial peptides
- Autor: Anna Diaz Cirac
- Universidad: Girona
- Fecha de lectura de la tesis: 01/07/2011
Dirección y tribunal
- Director de la tesis
- Pedro Salvador Sedano
- Tribunal
- Presidente del tribunal: María Joao ramos
- Manuel Nuno melo (vocal)
- paolo Carloni (vocal)
- lorenzo Stella (vocal)