Inhibiting Protein-Protein Interactions using Rationally-designed Repeat Proteins
The inhibition of protein-protein interactions (PPIs) presents a major challenge to drug discovery. The properties of consensus-designed tetratricopeptide repeat proteins (CTPRs) make them very amenable to be rationally-designed into potential new PPI inhibitors. This study sets out to investigate whether this could be achieved through the grafting of binding peptides into the inter-repeat loops. The work focused on two PPIs: the interaction between Kelch-like ECH-associated protein 1 (Keap1) and Nuclear factor erythroid-2-related factor (Nrf2) as well as the interaction between c-Myc and Myc-associated factor X (Max).
In Chapter 3, computationally-designed peptides were grafted into the inter-repeat loops of CTPRs as well as into another consensus-designed repeat protein, namely Designed Anrkyrin Repeat Proteins but no binding of these proteins to their target was observed. In Chapter 4, a number of Keap1-binding peptides were successfully grafted into the inter-repeat loop of a two-repeat CTPR2 protein, with the tightest binding functionalised CTPR having low nanomolar affinity for Keap1. In Chapter 5, new strategies were explored in order to further the understanding of how best to graft functional peptides into the inter-repeat loop of CTPRs, with the introduction of polyglycine residues either side of the grafted peptide being shown to lead to a greater than two-fold improvement in the binding affinity. In Chapter 6, it was shown that the functionalised CTPRs could be successfully delivered into HCT 116 by encapsulation within fusogenic liposomes. In Chapter 7, a peptide segment of a c-Myc inhibitor was grafted into a CTPR scaffold, and also engineered as a chemically stapled peptide. Preliminary pull-down experiments suggested that the stapled peptide bound to c-Myc in HeLa cell lysate.
This thesis demonstrates the amenability of the CTPRs to rational design with binding affinities in the low nanomolar range being obtained through peptide grafting into the inter-repeat loop. This highlights the potential application of this technology for the creation of future biotherapeutics.