Although numerous strategies for the identification of biologically active peptides are available, the methodologies to discover functional peptide pairs remain relatively scarce. The pairing of two combinatorial peptide libraries can furnish very large chemical spaces, which can be leveraged in ligand discovery to identify potent binders for proteins of interest. Here, we report the development of a library-vs-library in vitro selection platform for the discovery of heterodimeric macrocyclic peptide ligands. The platform is built upon the Random nonstandard Peptides Integrated Discovery (RaPID) system and utilizes protein-templated ligation of peptides from two mRNA display libraries to select for functional heterodimers. We report the method development and demonstrate the utility of the resulting protocols by identifying an ultrapotent heterodimeric ligand of 14–3–3ζ protein (h1.2, KD = 120 pM), which forms via a proximity-enabled inverse electron demand Diels–Alder reaction of two cyclic peptide monomers upon binding to the protein. The formation of h1.2 from a1 and b2 monomers was accelerated 560-fold in the presence of 14–3–3ζ, and the heterodimer had a superior affinity compared to the constituent monomers KD(a1) = 430 pM and KD(b2) = 700 pM. Our strategy may be useful in drug discovery to develop high-affinity ligands against oligomeric proteins, targets without prominent binding pockets, or for disrupting protein–protein interactions characterized by large interaction areas.
Ohno et al. (Tue,) studied this question.