Expanding Cell Free Biosynthesis for the Discovery of Natural Product-Inspired Peptides

Peptide-based drugs have historically been valuable assets in the clinic for combating disease. Traditionally, natural products have provided a rich source of carefully optimized molecules for peptide drug discovery efforts. However, advancements in de novo peptide screening through the advent of encoded library technologies, like mRNA display, have shifted the focus away from natural products. Although mRNA display has proven to be an effective tool for potent ligand discovery, there has been limited advancement of hits from these screens into the clinic, particularly for intracellular targets. This limitation arises from the inherent physicochemical properties of standard mRNA display libraries, which are often composed of large, hydrophilic peptides that are incapable of crossing biological membranes. Therefore, returning to natural products as a source of inspiration in mRNA display library design promises to unlock unexplored chemical space for the selection of peptide hits that are directly tractable in the clinic.Recently, there have been significant advances in the flexibility and power of in vitro cell-free translation systems. The increasing ability to incorporate non-canonical amino acids and complement translation with recombinant enzymes has enabled the cell-free production of peptide-based natural products and natural product-like molecules. Integrating these advances into mRNA display has the potential to furnish next-generation, natural product-inspired mRNA display libraries. Toward this goal, we first develop a novel peptide parsing algorithm that allows us to rapidly chart natural product chemical space, define the cell-free accessibility of this space, and expand the accessibility to a highly enriched natural product building block. Second, we investigate the catalytic mechanism of a macrocyclase enzyme involved in natural product biosynthesis, providing a foundation for future applications of this enzyme in peptide library modification. Finally, we develop and screen a natural product-inspired mRNA display library that significantly shifts the chemical space in line with more favorable physicochemical properties for clinical advancement. Overall, by combining the rapid screening capabilities of mRNA display with privileged scaffolds evolved in natural products, this work provides crucial advancements towards expanding the scope of peptide therapeutics.