Di Nardo, Ilaria (2024) Chemical strategies to improve proteolytical stability and biological activity of Host Defense Peptides. [Tesi di dottorato]

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Abstract

The rapid rise of drug-resistant infections poses a significant challenge to antimicrobial treatments. The inability of even the most powerful antibiotics to eradicate “superbugs” highlights an urgent need for alternative therapeutic options. Cationic antimicrobial peptides (CAMPs) are valid therapeutic alternatives to the use of conventional antibiotics in combating bacterial resistance, mainly due to their broad-spectrum antimicrobial activity, the limited ability of microorganisms to develop resistance and a multiplicity of different biological activities including immunomodulatory activity. However, the main disadvantage of these molecules lies in their protease sensitivity which limits their applicability. This thesis explores three different approaches to face the stability issue: (i) the development of a novel antimicrobial peptidomimetic; (ii) the design of bicyclic synthetic CAMPs; (iii) the production of multimers of recombinant CAMPs. The first of the three approaches resulted in the preparation of P13#1, a custom-designed 13-residue peptoid, with broad-spectrum bactericidal activity, LPS-scavenging ability similar to that of polymyxin E, antibiofilm activity greater than that of the human cathelicidin LL-37 and a promising antiviral activity against the porcine epidemic diarrhea virus (PEDV). Furthermore, it showed immunomodulatory and immunostimulatory effects in murine macrophages (Raw 264.7 cells) and murine dendritic cells (DC2.4). Furthermore, we developed bicyclic antimicrobial peptides obtained by chemically crosslinking with tris-(bromomethyl)benzene of linear synthetic peptides containing three cysteine residues. The cyclization procedure preserves the antimicrobial activity, the endotoxin scavenging activity and the low toxicity while significantly enhancing resistance to serum proteases. Finally, a strategy was developed to obtain multimers of recombinant CAMPs with a single terminal cysteine residue through crosslinking to multifunctional cysteine-reactive chemicals. The data reported in this thesis work shows that CAMP-like molecules resistant to proteases but endowed with all the pharmacologically desirable biological activities of the CAMPs can be obtained with different and complementary strategies each with its merits. Hopefully, the work reported in this PhD thesis will inspire the design of protease-resistant CAMPs and accelerate the pace of their clinical translation and their industrial exploitation.

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