Oliva, Rosario (2018) AMPs: Rational Design, Synthesis and Biophysical Studies of the Interaction Process with Model Membranes. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: AMPs: Rational Design, Synthesis and Biophysical Studies of the Interaction Process with Model Membranes
Oliva, Rosariorosario.oliva2@unina.it
Date: 11 December 2018
Number of Pages: 159
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
Paduano, Luigiluigi.paduano@unina.it
Petraccone, LuigiUNSPECIFIED
Del Vecchio, PompeaUNSPECIFIED
Date: 11 December 2018
Number of Pages: 159
Keywords: Peptides, Lipids, Biophysics
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/02 - Chimica fisica
Additional information: rosario.oliva11@gmail.com
Date Deposited: 19 Jan 2019 16:22
Last Modified: 22 Jun 2020 09:24
URI: http://www.fedoa.unina.it/id/eprint/12622

Collection description

The emergence of resistance from bacteria to the conventional antibiotics has become a serious global problem during the last years. The onset of resistance is mainly due to the massive and out-of-control use of these drugs in our community. In fact, microorganisms have developed a series of mechanism which render antibiotics ineffective. Therefore, seeking for new anti-infective agents is becoming increasingly necessary. Among these antimicrobial peptides (AMPs) have been proposed. AMPs are a class of peptides with a broad spectrum of activity against different pathogenic organisms (e.g. bacteria, fungi, viruses and cancer cells) and they are part of the innate immune system of virtually all forms of life. Usually, they are composed by 10-50 amino acids and are enriched of positively charged (e.g. lysine and arginine) and hydrophobic residues. Even if the exact action mechanism is still under debate, it is widely accepted that the primary target is represented by the lipid matrix of the pathogens’ membranes. Thus, AMPs interact with them in a non-specific way, inducing membrane destabilization and finally cell death. It is believed that, due to the unique mode of action, AMPs could overcome the problem of resistance to conventional antibiotics in multi-drug resistant bacteria and even to currently used anticancer drugs for the treatment of tumor cells. For these reasons, AMPs have attracted great attention as drugs of the future. Thus, the final goal in studying AMPs is their use as drugs. However, AMPs pharmacological application is not straightforward. Many aspects must be considered. A good AMP should interact selectively with the bacterial membrane, should not be toxic to eukaryotic cells and should be resistant to proteases degradation. To develop AMPs with these improved features it is of fundamental importance to understand the role played by lipid composition and peptide physico-chemical properties in determining peptides activity. To this aim, in my PhD thesis I studied the molecular details, at level of peptide-lipid interaction, of the action mechanism of several antimicrobial peptides by a series of biophysical techniques. In particular, I characterized the interaction of natural amino acids-containing peptide as well as of synthetic peptides composed by unnatural amino acids with model biomembranes. In the first part of the project, I carried out the synthesis of the 9-residue peptide P9Nal(SS) which contains unnatural amino acids. It was designed in order to obtain a peptide with a good antimicrobial activity, low cytotoxicity and high resistance to proteases. Then, biophysical studies of its interaction with eukaryotic and bacterial model membranes were carried out. The obtained information can be very useful in developing antimicrobial agents for biomedical applications. This study is described in detail in the chapter 3. In the chapter 4 is reported the biophysical characterization of the interaction of two P9Nal(SS)-derived peptides, named P9Nal(SR) and P9Trp(SS), with liposomes mimicking bacterial membrane. The two peptides were obtained by replacing some residues in the primary sequence of P9Nal(SS). As shown in chapter 4, the peptides are less hydrophobic than the parent peptide. The obtained data reveal how these substitutions can modulate the membranotropic activity of peptides. In the second part of the project, I faced the problem to understand the molecular details of the action mechanism of (P)GKY20, a natural amino acids-containing peptide modelled on the C-terminus region of the human thrombin. Its good antimicrobial activity and low cytotoxicity is well known. However, its action mechanism has never been studied before. Thus, I performed an extensive biophysical characterization of the interaction of the (P)GKY20 with model membranes. The obtained results elucidated its mechanism of action against bacterial model membrane and, at the same time, allow to understand its low cytotoxicity. All the results concerning this AMP are presented in the chapter 5. The last part of this thesis is devoted to an idea developed during the third year: the encapsulation of AMPs with cyclodextrins (CDs) as a way to protect the peptides from degradation and improve their pharmacological properties. Indeed, antimicrobial peptides containing natural L amino acids are, unfortunately, prone to degradation which limits seriously AMPs applications as drugs. To verify the ability of CDs to encapsulate AMPs without altering their antimicrobial properties, I studied the interaction of (P)GKY20 peptide with sulfobutylether-β-CD and of the obtained complex with bacterial-like liposomes. These preliminary results reveal that (P)GKY20 form a 1:1 stable complex with sulfobutylether-β-CD. Further, the obtained complex is able to perturb the stability of bacterial-like liposomes. Thus, the sulfobutylether CD could represent a suitable encapsulating agent for (P)GKY20 peptide which could improve its pharmacological profile. The obtained results are presented in the chapter 6. Since the peptide interaction with model membranes is quite complex being composed by many steps, a strategy which involves different biophysical techniques was adopted to study a particular aspect of the interaction process. Calorimetric, spectroscopic and microscopic techniques were applied (where possible) and by combining the data, it was possible to depict a possible action mechanism highlighting the key steps. Differential Scanning Calorimetry (DSC) was employed to study the effect of peptide interaction on the liposomes thermotropic properties. Moreover, using an appropriate lipid system, information about lipid self-organization (e.g. lipid domains formation) can be obtained. Steady-State Fluorescence Spectroscopy and Fluorescence Anisotropy were extensively employed to obtain information on both AMPs (e.g. determination of binding constants, degree of insertion into the lipid bilayer) and lipids (e.g. changes in membrane viscosity due to changes in lipid packing). Liposomes morphological changes were monitored by Dynamic Light Scattering (DLS). Instead, changes in peptide secondary structure upon binding were followed by means of Circular Dichroism (CD) and an estimation of secondary structure content was obtained by the spectra deconvolution. Atomic Force Microscopy (AFM) and Confocal Fluorescence Microscopy instruments were employed to directly visualize the effect of peptides on bacterial model membranes.


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