Grafted Coiled-Coil Peptides as Multivalent Scaffolds for Protein Recognition

Self-assembled peptides are promising templates for the design of inhibitors of protein-protein interactions (PPIs) because they can be endowed with affinity- and selectivity-defining amino acids alongside favorable physicochemical properties such as solubility and stability. Here, we describe a tunable coiled-coil scaffold and its interaction with MCL-1, an α-helix-binding antiapoptotic protein and important target in oncology. We explore the role of oligomerization, multivalency, and cooperativity in PPI inhibition. Hot-spot residues from an MCL-1 binding peptide (NOXA-B) are grafted onto the outer surfaces of homo- and heterodimeric coiled-coil peptides to obtain inhibitors with mid-nM potency and selectivity over BCL-x_L. Binding of homodimeric coiled coils to MCL-1 is positively cooperative, resulting in stabilization of both partners. Homodimeric coiled coils support the binding of two copies of the target protein. Modification of the coiled-coil sequence to favor assembly of higher-order scaffolds (trimer and tetramer) negatively impacts inhibitory potency, with AlphaFold2 modeling and biophysical data indicating a complex interplay between coiled-coil oligomerization and target binding. Together, these data establish dimeric coiled coils as the most promising of such scaffolds to develop inhibitors of α-helix-mediated PPIs.