Dottorato in MODELLI MATEMATICI PER L'INGEGNERIA, ELETTROMAGNETISMO E
NANOSCIENZE

Titolo della tesi: Bile acids-based antimicrobial peptides

ABSTRACT: Antimicrobial resistance represents an increasing threat to public health and makes urgent the need for new-generation antimicrobials with novel modes of action and potent therapeutic activity. Natural antimicrobial peptides (AMPs) have been widely regarded as a promising solution to combat multi-drug resistant bacteria, due to their broad-spectral bactericidal activity, rapid killing rate, and a membrane targeted mechanism of action that gives them low susceptibility for developing resistance. Typically, AMPs are ∼12-50 amino acid residues in length, have net positive charge, and contain around 50% hydrophobic amino acid residues. They adopt a facial amphiphilic structure when in contact with bacterial membranes, by arranging positive charges and lipophilic groups on opposite sides. Positive charge and facial amphiphilic structure are considered essential for AMPs to bind to the negatively charged bacterial membranes and penetrate inside them leading to cell death. However, high manufacturing cost, susceptibility to proteolytic degradation, nonspecific toxicity to host cells and limited bioavailability hinder the application of AMPs in clinic [1]. Bile acids (BAs) [2], a group of cholesterol-derived steroid acids produced in mammals and other vertebrates, with high stability, biocompatibility, and inherent facial amphiphilic structure, have been exploited as substrate to prepare a new class of steroid antibiotics. In fact, they are able to mimic the AMPs characteristics of positive net charge, facial amphiphilicity and a proper hydrophobic/hydrophilic balance. In this work we present the synthesis, physical-chemical characterization, antimicrobial activity and cytotoxicity studies of a new class of BAs-based antimicrobials, obtained by joining the facial amphiphilic skeletons of BAs to cationic peptides. The incorporation of some D-amino acids is a traditional strategy for creating protease-resistant antimicrobial peptides. We decided to examine the effects of regularly alternating L,D-amino acids motif, since previous studies showed peculiar self-assembling behaviour of such configurationally alternating sequences that could enhance the molecule antimicrobial efficacy [3]. We obtained these unconventional peptide amphiphiles (PAs) by the conjugation of a peptide chain to the carboxylic group of a BA molecule through a peptide bond. This was achieved by solid phase peptide synthesis, a relatively simple procedure with high yields. The physical-chemical properties of these BAs-based PAs were investigated through multiple experimental techniques, such as Dynamic Light Scattering, Electrophoretic Mobility, Circular Dichroism, proton Nuclear Magnetic Resonance, Small-Angle X-ray Scattering and Fluorescence Spectroscopy. Quartz Crystal Microbalance with Dissipation was used to study the absorption and interaction behaviour of the BAs-based PAs with cell membrane models, consisting of phospholipid bilayers supported on polyelectrolyte multilayers cushion. The antimicrobial activity was tested against model bacteria such as Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, with very promising results. REFERENCES: [1] M. Pasupuleti, A. Schmidtchen and M. Malmsten, Crit. Rev. Biotechnol., 2012, 32, 143–171 [2] M. C. di Gregorio, L. Travaglini, A. del Giudice, J. Cautela, N. V. Pavel and L. Galantini, Langmuir, 2019, 35, 6803-6821 [3] F. Novelli, S. De Santis, S. Morosetti, M. Titubante, G. Masci, A. Scipioni, Peptide Science, 2018; 110:e24043