Modulating Protein-Protein Interactions via Peptide-Based Inhibitors: Structural and Functional Insights

As potential therapeutic targets, protein-protein interactions (PPI) are primary to cellular function and processes. This thesis explores peptide-based PPI inhibitors with respect to their design, structural characterization, and functional analysis. The main focus is the modulation of peptide-based PPI inhibitors through conformational stabilization and binding affinity optimization. Chapter 1 (Introduction) provides an overview of protein structure principles, highlighting the importance and role of PPI in biological processes. It introduces the challenges related to targeting these interactions and the ability of peptides and peptidomimetics as promising PPI modulation tools. Furthermore, background information of structural biology techniques such as X-ray crystallography, and biophysical methods is provided. Chapter 2 (Peptide-Based Covalent Inhibitors of Protein-Protein Interactions) reviews strategies to stabilize protein structures and the progression from peptides to peptidomimetics. One of the key focuses is the importance of structure stabilization to design functional biomimetics. It also covers how stabilization of protein structures can increase their usefulness in biotechnological applications. Chapter 3 (Fragment Screening of NF-YB/C Heterodimer: Towards Novel Inhibitors of NF-Y Activity) investigates the identification of potential small-molecule inhibitors of the NF-Y trimer formation using fragment-based drug design approach. An attempt was made to optimize crystallization system for successful fragment screening, thus crystallization optimizations were carried out, nonetheless no fragment hits were acquired. Hence, this chapter also discusses the difficulties in targeting NF-Y with fragment-based discovery, and shares insights that might be useful for alternative screening and inhibitor design strategies. Chapter 4 (Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y) researches the design, synthesis, and characterization of different stapled peptides as NF-Y inhibitors. Hydrocarbon peptide stapling improves peptide helicities and interaction of the peptides with the protein target. Moreover, this chapter identifies the minimal binding core in a 13-mer stapled peptide and discovers the flexibility of its structure when bound to NF-YB/C heterodimer. Chapter 5 (Helicity-Dependent Enzymatic Peptide Cyclization) describes how peptide α-helicity influences enzymatic head-to-tail peptide cyclization using engineered Sortase. Results show that peptides with low helicity allow efficient cyclization, while more rigid helical peptides incline towards complex cyclization by forming cyclic dimers or oligomers. This chapter also features how fine-tuning secondary structures can influence the chemical reaction through modulating the relationship between peptide rigidity and enzymatic cyclization.