Pane, Katia (2016) Development and optimization of topical antimicrobial agents based on human antimicrobial peptides. [Tesi di dottorato]

Preview

Text PANE_KATIA_28.pdf Download (2MB) | Preview

Collection description

New antimicrobial compounds are urgently needed to fight infective diseases caused by antibiotic-resistant human pathogens. AntiMicrobial Peptides (AMPs) are attractive candidates as they can both directly kill microbes and modulate immune response. Surprisingly, many human proteins whose functions are not necessarily related to host defense can behave as sources of AMPs. Some examples are lactoferrin, lysozyme or thrombin. Since these AMPs are hidden in large proteins, they can be defined “cryptic”. In order to identify by a rational approach further human proteins carrying new cryptic AMPs, a scoring function allowing a quantitative prediction of antibacterial activity was developed and validated analyzing the sequence of already known AMP releasing proteins. Using this method to screen about 4'000 human extracellular proteins, a wide list of potential new AMPs was obtained. The main aim of the present PhD thesis is to select and characterize the most pharmacologically interesting potential AMPs in order to find candidates suited for the development of novel human antimicrobial and anti-inflammatory agents. Firstly, I developed a novel cost-effective fusion system, particularly well-suited for the production of toxic peptides in E. coli. To avoid AMPs toxicity toward bacterial host, I rationally designed a carrier protein starting from a Rana pipiens ribonuclease, named onconase (ONC), that is expressed at high level (200 mg/L of culture) as inclusion bodies in E. coli. In order to optimize this method, a well-known AMP derived from human thrombin, named GKY20 was used as model peptide. Moreover, I demonstrated that this strategy also allows to prepare peptides with a N-terminal cysteine residues that could find many applications in basic and applied research like for example preparation of labeled peptides, protein ligation for the semi-synthesis of modified proteins, immobilization on supports etc. Using this procedure I prepared and characterized several new human AMPs from 20 to almost 50 amino acids with yields of at least 10 mg/L of culture. Therefore, our method is complementary to chemical synthesis being particularly well-suited to prepare peptide longer than 20-25 residues. All these peptides show a broad antimicrobial activity on GRAM-negative and GRAM-positive bacteria, antibiofilm and immunomodulatory activity. Very interestingly, they proved to be both non-toxic on eukaryotic cells, providing AMPs of human origin particularly well suited to develop anti-infective drugs.

Downloads

Actions (login required)

View Item