Late-Stage Reshaping\nof Phage-Displayed Libraries\nto Macrocyclic and Bicyclic Landscapes using a Multipurpose Linchpin

Genetically encoded libraries (GEL) are increasingly\nbeing used\nfor the discovery of ligands for “undruggable” targets\nthat cannot be addressed with small molecules. Foundational GEL platforms\nlike phage-, yeast-, ribosome-, and mRNA-display have enabled the\ndisplay of libraries composed of 20 natural amino acids (20AA). Unnatural\namino acids (UAA) and chemical post-translational modification (cPTM)\nexpanded GEL beyond the 20AA space to yield unnatural linear, cyclic,\nand bicyclic peptides. The standard operating procedure incorporates\nUAA and cPTM into a “naive” library with 10<sup>8</sup>–10<sup>12</sup> compounds and uses a chemically upgraded\nlibrary in multiple rounds of selection to discover target-binding\nhits. However, such an approach uses zero knowledge of natural peptide-receptor\ninteractions that might have been discovered in selections performed\nwith 20AA libraries. There is currently no consensus regarding whether\n“zero-knowledge” naive libraries or libraries with pre-existing\nknowledge can offer a more effective path to discovery of molecular\ninteractions. In this manuscript, we evaluated the feasibility of\ndiscovery of macrocyclic and bicyclic peptides from “nonzero-knowledge”\nlibraries. We approach this problem by late-stage chemical reshaping\nof a preselected phage-displayed landscape of 20AA binders to NS3aH1\nprotease. The reshaping is performed using a novel multifunctional\nC<sub>2</sub>-symmetric linchpin, 3,5-bis­(bromomethyl)­benzaldehyde\n(termed KYL), that combines two electrophiles that react with thiols\nand an aldehyde group that reacts with N-terminal amine. KYL diversified\nphage-displayed peptides into bicyclic architectures and delineated\n2 distinct sequence populations: (i) peptides with the HXDMT motif\nthat retained binding upon bicyclization and (ii) peptides without\nthe HXDMT motif that lost binding once chemically modified. The same\nHXDMT family can be found in traditional selections starting from\nthe naive KYL-modified library. Our report provides a case study for\ndiscovering advanced, chemically upgraded macrocycles and bicycles\nfrom libraries with pre-existing knowledge. The results imply that\nother selection campaigns completed in 20AA space, potentially, can\nserve for late-stage reshaping and as a starting point for the discovery\nof advanced peptide-derived ligands.