USING B-STRAND AND B-HAIRPIN PEPTIDOMIMETICS INDUCED BY CONSTRAINED AMINO ACIDS TO MODULATE THE AGGREGATION OF THE MICROTUBULE ASSOCIATED PROTEIN TAU: DESIGN, SYNTHESIS AND EVALUATION

Tauopathies refer to neurodegenerative disorders characterized by the presence of abnormal aggregates of Tau protein, leading to the formation of neurofibrillary tangles (NFTs) within neuronal cells. Tau, a protein associated with microtubules (MTs), plays a crucial role in regulating the dynamics of MTs and facilitating proper axonal transport in neurons. Alzheimer's disease (AD) is characterized by abnormal protein-protein interactions (PPIs) which lead to the accumulation of β-amyloid plaques and hyperphosphorylated Tau (P-tau) NFTs. Multiple evidence has suggested that the interplay between Tau and Aβ contribute to worsening the disease since the aggregation of one protein can trigger the aggregation of the other. Within this thesis, we have explored, for the first time, the possibility of creating peptidomimetics capable of stabilizing either a β-hairpin or an extended structure to modulate Tau protein aggregation. This work focuses on three distinct peptidomimetic units with the goal of highlighting the significant role of secondary structure in the selective regulation of both physiological and pathological PPIs of Tau. The utilization of a previously developed piperidine-pyrroline β-turn mimic has led to highly effective peptidomimetics that act as dual inhibitors targeting both Tau and Aβ1-42 proteins but show no activity on the other amyloid protein hIAPP. These compounds were designed either on Tau PPIs, by mimicking its self-interaction in NFTs or its physiological interaction with MTs; or on small sequences of the chaperone protein Hsp90, which is a physiological Tau aggregation inhibitor: Through various analyses, including proteolytic stability, ThT fluorescence, cell viability (LDH and MTT), and live cell imaging FDAP assays, we have demonstrated the critical role of pre-organized hairpin structures in achieving potent activity, selectivity, and stability. Among the designed compounds, the one mimicking Hsp90-Tau- interaction showed the highest potency, and NMR titration with 15N-Tau protein provided valuable insights into the possible mechanism of action of Hsp90 on Tau. Then, the employment of the non-natural β2,2-isoxazoline unit into a peptide sequence was used to stabilize two different secondary structures, either in β-hairpin or extended. These structures were confirmed through a combination of CD, IR, and NMR analyses in aqueous solvents. The stabilized hairpin structure was used to develop our most potent inhibitor of Tau aggregation by mimicking Hsp90 protein. The extended structure bearing the PHF6 and PHF6* sequences (both involved in the physiological and pathological processes of Tau), was used to prove that it is the exposure of these sequences on Tau protein the event triggering its aggregation. As a counter-test, the hairpin conformation with the same sequences, did not induce any pathological aggregation. Finally, the development of new difluorinated triazoles and their incorporation into existing peptide inhibitors of Tau, led to enhance their effectiveness, as demonstrated by ThT and live cell FDAP imaging, possibly because of their increased pharmacokinetic properties driven by the presence of the fluorine atoms. In summary, the findings presented in this thesis provide a proof of concept that pre-structured hairpin mimics serve as a solid foundation for the development of innovative inhibitors of Tau and Aβ aggregation. The chaperone mimicry strategy has demonstrated its remarkable efficacy being the most potent dual inhibitor and, providing at the same time a new probe to simplify and study Tau-Hsp90 physiological interactions. The inclusion of the β2,2-isoxazoline amino acid into the peptide sequence allowed to demonstrate that the conversion of Tau toward an extended conformation, with the consequent exposure of PHF6* and PHF6, is the triggering event leading to Tau self-assembly.