Nonproteinogenic deep mutational scanning of linear and cyclic peptides

Abstract

SignificanceThe 20 proteinogenic amino acids have physicochemical properties that allow peptides and proteins to fold and bind. However, there are numerous unnatural, nonproteinogenic amino acids that may be equally good, or even better, at folding and binding. Exploration of these alternative peptide building blocks has been limited by slow, one-at-a-time synthesis and testing. We describe how, in a single experiment, multiple nonproteinogenic amino acids can be trialed at all positions in a peptide sequence, with thousands of modifications tested in parallel. This permits detailed analysis of how chemical structure relates to function and allows for systematic comparisons of proteinogenic and nonproteinogenic chemistry. Such analysis can guide the improvement of drug-candidate peptides, including the therapeutically promising class of cyclic peptides.

Variant effect predictions capture some aspects of deep mutational scanning experiments

“…DMS also screens proteins for improved drug binding, antibody affinity, using non-native chemical stresses, or non-proteinogenic amino acids, and on synthetic proteins [19][20][21][22][23][24][25][26]. Finally, DMS share objectives with directed evolution, benefiting protein engineering [14].…”

Ultra-large chemical libraries for the discovery of high-affinity peptide binders

“…Peptides are synthetically accessible, amenable to chemical tailoring, and have the potential to bind the typically shallow surfaces seen in therapeutically relevant-and historically intractable-protein-protein interactions (PPIs) [6][7][8] . Importantly, chemical modifications, such as non-canonical amino acid incorporation, head-to-tail macrocyclization, and chemical stapling, can render peptides more proteolytically stable, more cellpenetrant, and even increase binding affinity relative to their natural, underivatized counterparts, which on their own tend to exhibit poor pharmacological properties [9][10][11][12][13] .…”

Peptide Folding and Binding Probed by Systematic Non-canonical Mutagenesis

“…Mutated amide oxygens shown as spheres and colored according to G. (E) Deep mutagenesis scanning (DMS) allows thousands of mutations to be collected, and saturation mutagenesis to be performed. Shown is the saturation mutagenesis data for PUMA binding MCL-1 (Rogers et al, 2018). (F) Slice of DMS data for the folded YAP65 WW domain binding its peptide ligand; Ala mutations colored according to DMS enrichment score where negative score indicates weaker binding (Fowler et al, 2010) (PDB 1JMQ).…”

“…(A) Reprogramming of ribosomal peptide synthesis allows deep mutational scanning to been expanded to include mutations to non-canonical amino acids. Non-canonical scanning (NCS) data for PUMA shown, with a selection of the non-canonical amino acids tested (Rogers et al, 2018). (B) Slices of NCS data for PUMA binding and scans with D-stereochemistry alanine (DAl) and cyclohexyl-alanine (Cha), side-chains colored according to G. (C) Cyclic peptides are a promising new modality due to their impressive protein binding abilities.…”