Grafted Coiled-coil Peptides as Multivalent Scaffolds for Protein Recognition

Self-assembled peptides are promising templates for the design of inhibitors of protein-protein interaction (PPI) 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 anti-apoptotic protein and important target in oncology. We explore the role of oligomerization, multivalency and co-operativity 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-xL. Binding of homodimeric coiled coils to MCL-1 is positively co-operative resulting in stabilization of both partners. Homodimeric coiled coils support 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 modelling 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.