Late-stage reshaping of phage-displayed libraries to macrocyclic and bicyclic landscapes using multipurpose linchpin

Genetically-encoded libraries (GEL) are increasingly used for discovery of ligands for ‘undruggable’ targets that cannot be addressed with small molecules. Foundational GEL platforms like phage-, yeast-, ribosome- and mRNA-display enabled display of libraries composed of 20 natural amino acids (20AA). Today, numerous strategies expand GEL beyond 20AA space by incorporating unnatural amino acids (UAA) and chemical post-translational modification (cPTM) to build linear, cyclic, and bicyclic peptides. The standard operating procedure for UAA and cPTM libraries starts from a "naïve" chemically-upgraded library with 108-1012 compounds, uses target of interest and rounds of selection to narrow down to a set of receptor binding hits. However, such approach uses zero knowledge of natural peptide-receptor interactions which already exists in libraries with 20AA space. There is currently no consensus whether ‘zero knowledge’ naïve libraries or libraries with pre-existing knowledge can offer a more effective path to discovery of molecular interactions. In this manuscript, we evaluated the feasibility of discovery of macrocyclic and bicyclic peptide from "non-zero knowledge" libraries. We approach this problem by late-stage chemical reshaping of phage-displayed landscape of 20AA binders to NS3aH1 protease. The re-shaping is performed under a novel multifunctional C2-symmetric linchpin, 3,5-bis(bromomethyl)benzaldehyde (termed KYL), that combines two electrophiles that react with thiols and aldehyde group that reacts with N-terminal amine. KYL diversified phage-displayed peptides into bicyclic architectures and delineates 2 distinct sequence populations: (i) peptides that retained binding upon bicyclization (ii) peptides that lost binding once chemically modified. Our report provides a case study for discovering advanced, chemically-upgraded macrocycles and bicycles from libraries with pre-existing knowledge. The results imply that thousands of selection campaigns completed in 20AA space, in principle, can serve for late-stage reshaping and as a starting point for discovery of advanced peptide-derived ligands.