Targeting Protein Interaction Hotspots Using Structured and Disordered Chimeric Peptide Inhibitors

The main challenge in inhibiting protein-protein interactions (PPI) for therapeutic purposes is designing molecules that bind specifically to the interaction hotspots. Adding to the complexity, such hotspots can be within both structured and disordered interaction interfaces. To address this, we present a strategy for inhibiting the structured and disordered hotspots of interactions using chimeric peptides that contain both structured and disordered parts. The chimeric peptides we developed are comprised of a cyclic structured part and a disordered part, which target both disordered and structured hotspots. We demonstrate our approach by developing peptide inhibitors for the interactions of the antiapoptotic iASPP protein. First, we developed a structured, α-helical stapled peptide inhibitor, derived from the N-Terminal domain of MDM2. The peptide bound two hotspots on iASPP at the low micromolar range and had a cytotoxic effect on A2780 cancer cells with a half-maximal inhibitory concentration (IC₅₀) value of 10 ± 1 μM. We then developed chimeric peptides comprising the structured stapled helical peptide and the disordered p53-derived LinkTer peptide that we previously showed to inhibit iASPP by targeting its disordered RT loop. The chimeric peptide targeted both structured and disordered domains in iASPP with higher affinity compared to the individual structured and disordered peptides and caused cancer cell death. Our strategy overcomes the inherent difficulty in inhibiting the interactions of proteins that possess structured and disordered regions. It does so by using chimeric peptides derived from different interaction partners that together target a much wider interface covering both the structured and disordered domains. This paves the way for developing such inhibitors for therapeutic purposes.