Project Summary Most drugs target proteins with relatively deep pockets, such as enzyme active sites. However, screening collections comprised of these traditional Rule of 5-compliant molecules have consistently failed to provide high quality drug leads for many targets that function via protein-nucleic acid and protein-protein interactions (PPIs). These are the “difficult” or “undruggable” protein targets, which by some estimates comprise up to 85% of the human proteome. The importance of these types of PPIs has been well noted in cancer therapeutics and more recently in the infectious diseases field where PPIs play a role in the interaction between host and pathogen. Macrocyclic peptides (MPs) are capable of engaging these shallow, solvent exposed surfaces, but most MPs are not cell permeable. This highlights the critical need for the development of libraries of novel, cell permeable compounds capable of engaging PPI surfaces. In Phase I, we successfully demonstrated the feasibility of generating and screening large DNA-encoded libraries of non-peptidic macrocycles, which have the appropriate characteristics to address this need. In this Phase II project, we will expand the chemical diversity of these libraries, test them against several difficult targets, and establish methods to evolve primary screening hits derived from the libraries into high-quality drug leads.