MECHANISM OF ACTION OF CYCLIC ANTIMICROBIAL PEPTIDES Anna DÍAZ i CIRAC Dipòsit legal: GI-1303-2011 http://hdl.handle.net/10803/38252 ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. 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PhD Thesis Mechanism of Action of Cyclic Antimicrobial Peptides Anna D´ıaz Cirac 2011 Doctorat Interuniversitari en Qu´ımicaTe`oricaComputacional Dirigida per Pedro Salvador Sedano Marta Planas Grabuleda Lidia Feliu Soley Mem`oriapresentada per optar al t´ıtolde Doctor per la Universitat de Girona Pedro Salvador Sedano, Marta Planas Grabuleda i Lidia Feliu Soley profes- sors titulars del Departament de Qu´ımicade la Universitat de Girona, CERTIFIQUEM: Que aquest treball titulat “Mechanism of Action of Cyclic Antimicro- bial Peptides”, que presenta l’Anna D´ıazCirac per a l’obtenci´odel t´ıtolde Doctor, ha estat realitzat sota la nostra direcci´oi que compleix els requeriments per poder optar a Menci´oEuropea. Signatura Pedro Salvador Sedano Marta Planas Grabuleda Lidia Feliu Soley Girona, 26 d’Abril de 2011 iii A la meva mare Summary of the Thesis 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 vesicato- ria, 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 candi- dates 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 computa- tional 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 re- placing the phenylalanine residue by a tryptophan for the cyclic peptides from the library is evaluated in detail. Even though the tryptophan analogue shows vii 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 sev- eral specific cyclic AMPs from the library. In a first study, we describe a compre- hensive 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 com- pact 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 β-turn I and α-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. Fur- thermore, 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 β-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 simula- tions 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 fun- damental 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. Princi- pally based on the bioactive conformation of BPC194, nine cyclic peptides were designed and synthesized. Remarkably, peptide BPC490 exhibits improved activ- ity against all three bacteria compared to the most active peptide of the original library, yet keeping its hemolytic activity still very low. Such high antimicro- bial activity found for some of these new peptides evidences the effectiveness of our rational design approach, by combining both experiment and computational modeling. Resum de la Tesi Els p`eptids antimicrobians (AMPs) es troben actualment en el centre d’atenci´ocom a possibles candidats per superar la resist`enciabacteriana als antibi`oticsconvencionals. El mecanisme pel qual aquestes mol`eculesmaten els bacteris per interacci´oamb la membrana cel.lular no est`acompletament ent`es. L’estudi dels processos biof´ısicsde l’activitat antimicrobiana d’aquests p`eptids poden proporcionar informaci´orellevant per al disseny racional de nous candi- dats per al millor desenvolupament d’efica¸cosagents antibacterians. D’entre el gran nombre de AMPs presents en la natura, els p`eptidsc´ıclicss’han convertit en bons candidats a compostos antimicrobians a causa de la seva robusta estructura secund`ariai la seva elevada activitat. El punt de partida d’aquesta tesi ´esuna biblioteca de de novo decap`eptids c´ıclics sintetitzat al laboratori LIPPSO de la Universitat de Girona, que va mostrar una elevada activitat antimicrobiana contra tres bacteris pat`ogensde plantes (Pseudomonas syringae, Erwinia amylovora i Xanthomonas vesicatoria), aix´ıcom una activitat hemol´ıticabaixa. L’estructura prim`ariad’aquests p`eptids c´ıclicsalternava amino`acidscati`onics(lisina) i hidrof`obiques(leucina i fenilalan- ina), per donar lloc a la f´ormula general c(X5-Phe-X3-Gln), on X poden ser lisines o leucines.