BIOPHYSICAL CHARACTERIZATION OF A NOVEL CLASS OF MEMBRANE-ACTIVE BRANCHED POLYPEPTIDES

Ever increasing number of resistant pathogens is seriously threatening global health care. New classes of antimicrobials and alternative strategies are urgently needed. Biologically active molecules can be rationally modified in order to tailor their activity for specific therapeutic applications. Among different new molecular classes investigated during the last decades, antimicrobial peptides have received particular attention. However, beside great potentialities and versatility, severe drawbacks limit their actual application and developments as drugs. Usually, high activity is accompanied by not negligible toxicity. Antimicrobial peptides typically exploit their activity by targeting and altering the fundamental physical properties of biological membranes. Although numerous synthetic and semi-synthetic strategies have met success in solving a variety of their drawbacks, the new class of direct microbicidal drugs has still to come. As the World Health Organization suggested, we should also think about new adjuvants to help the already available antimicrobial drugs to overcome their current limitations in tackling resistant pathogens. The present work was devoted to the characterization of a novel class of branched polypeptides, endowed with several advantages over typical natural antimicrobial peptides, like the lower susceptibility to proteases degradation. A lysine linker represented the core of the dendrimeric architecture, with two identical copies of the active 10-mer amino acid sequence bound on the α- and ε-nitrogen, respectively, and an octanamide tail at the C-terminus. In recent years, the research group has provided first fundamental information such as antimicrobial activity in standard media, qualitative estimation of bilayer affinity and 3D structure in simple membrane mimicking environments. An intensive biophysical characterization has been undertaken during the last three years within the present PhD programme, aimed at more comprehensive understanding of the behaviour of such unusual peptides. Particular efforts were spent to unveil the mechanism of action, a difficult task that required combination of different methodologies. Spectroscopic techniques, like both absorbance and fluorescence UV-vis spectroscopy and attenuated total reflectance infrared spectroscopy represented the main investigation tools, together with surface pressure measurements of lipid monolayers at the air-water interface. In addition to dynamic light scattering, setup of numerical models to analyse the results completed the strategy. Different collaborations were also needed to provide further insight from microbiological assays and transmission electron microscopy. The first aim of this work was the comparison of two analogues with the same tail and net positive charge, allows focusing on the effects of amphipathic profile regularization of the amino acid sequence. Overall, the investigated peptides were found to fold in β-hairpin like conformation at a monomeric level, and to form extended aggregations with increasing peptide local concentration on the membrane. The structural order was determined by the inherent characteristics of the peptide sequence as well as by system electrostatics, ultimately affecting peptide activity. The second aim focused on several analogues with highly hydrophobic tail of different length. Quite unexpectedly, a membrane-anchoring tail worsened peptide efficacy. Structure-function investigations on dendrimenric peptides are almost completely lacking. It is fundamental to fill in this gap and to understand what is really due to the specific peptide sequence, to the branching spacers or to the branches number, if rational design of such complex molecular architecture is the goal. In addition, the characteristic β-type folding makes the investigated peptides interesting as elements of a structural class of membrane-active peptides that has still to be fully understood.