Campanile, Marco (2024) Mechanism of action of therapeutic peptides: from membranes to intracellular targets. [Tesi di dottorato]

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Abstract

Therapeutic peptides have emerged as promising candidates to combat diseases such as cancer and viral infections. Among them, host defense peptides (HDPs) are evolutionary conserved molecules with anticancer, antimicrobial and antiviral properties. This PhD thesis focuses on non-membranolytic HDPs, particularly LL-III, and their ability to interact with both cellular membranes and intracellular targets. Using biophysical techniques, LL-III was shown to transiently destabilize negatively charged model tumor membranes, enabling its non-disruptive internalization. The peptide was then tested against biologically relevant DNA G-quadruplexes as possible intracellular targets and was found to exhibit a nanomolar affinity despite the lack of common G-quadruplex binding motifs. To establish whether LL-III can also recognize non-canonical RNA structures, a sequence from the SARS-CoV-2 genome, RG1, was selected. The RG1 was shown to adopt a G-triplex conformation rather than the previously assumed G-quadruplex at physiological temperature. Preliminary data indicated that LL-III can successfully recognize RG1 under these conditions, suggesting that this peptide may also hold potential as an antiviral agent. Finally, LL-III’s effects on a model SARS-CoV-2 condensate containing the nucleocapsid protein were assessed, showing that the peptide can alter the shape and dynamics of the viral condensates, with possible implications the viral replication. Comparative studies with other HDPs provided a first glimpse into the role of peptide physicochemical properties in biomolecular condensates modulation. This work underscores the broader therapeutic potential of non-membranolytic HDPs, paving the way for innovative and effective multi-target therapeutic strategies.

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