Barone, Simona (2025) Development of epigenetic modulators. [Tesi di dottorato]

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Abstract

This PhD thesis aimed at the identification of novel epigenetic modulators to address major health burden diseases, including oncological and infectious diseases, inflammatory states related to persistent bacterial and fungal infections, and rare diseases. In this context, compelling new evidence linked histone deacetylase 6 (HDAC6) activity to a wide range of biological processes, including cell cycle regulation, apoptotic and autophagic events, stress response and protein quality control, immune system modulation and cytoskeletal dynamics. Accordingly, HDAC6 aberrant activity or expression has been linked to the onset and progression of several pathological states, including cancer, rare diseases and inflammatory disorders. Moreover, unlike other members of HDAC family, HDAC6 presents unique structural features (e.g. the presence of two tandem catalytic domains) and predominantly cytoplasmic localization, enabling it to exert its activity on non-histone proteins (e.g. tubulin, HSPs, ubiquitin, cortactin) involved in key physiological and pathological processes. Thereby, selectively targeting HDAC6 is emerging as a promising strategy for the treatment of pathological states with a strong epigenetic component driven by HDAC6, while minimizing side effects related to pan HDAC inhibition. Accordingly, during my PhD I was involved in the design of novel spiro-capped HDAC6 inhibitors, in order to both improve selectivity and potency – in comparison to available inhibitors - while improving pharmacokinetic properties and safety profile. Driven by in silico studies, we first identified 1,3,8- triazaspiro[4,5]decan-4-one as a suitable capping group for preferential interaction within HDAC6 catalytic surface, while the canonical hydroxamic acid was initially selected as the zinc binding group (ZBG). All synthesised compounds displayed high potency towards HDAC6 and high selectivity over HDAC1 and HDAC8 (as representative of other HDAC classes), while showing efficacy in activating both pro- apoptotic and autophagic pathways, as well as inhibiting cell migration and epithelial-to mesenchymal transition (EMT) in both monolayer and 3D cellular cultures (spheroids) of MDA-MB-231 triple negative breast cancer (TNBC) cells. Starting from these promising results, we sought to perform a linker inversion on the newly conceived cap group, in order to exploit the different cap group geometry to enhance binding affinity within HDAC6, also by targeting uncommon biding sites on the enzyme. During the second year of my PhD, I had the opportunity to spend six months in Finn K. Hansen research group (University of Bonn) working on the introduction of TMFOs and DFMOs ZBGs on a newly conceived 1-thia-4,8-diazaspiro[4.5]decan-3-one cap group. Moreover, proprietary selective HDAC6is presented in this thesis work showed efficacy in modulating P. aeruginosa and C. albicans virulence, thereby offering a new glimpse in the treatment of resistant bacterial and fungal infections. In parallel, in the field of infectious diseases, I took part in the design and synthesis of novel pyrazole-isoxazole antifungal agents in synergy with voriconazole in C. albicans strains resistant to voriconazole itself.

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