Development and Advancement of Phage Display Techniques for the Identification of Therapeutic Peptides

Abstract

Peptides represent an emerging class for drug discovery, with the number of therapeutic peptides nearly doubling in the past twenty years. In spite of the development of many different screening techniques to identify potentially therapeutic peptides for new drug targets, the majority of these display peptides that have low therapeutic potential due to poor pharmacokinetics. One of the most prominent methods for screening large peptide libraries that has developed over the past thirty years is phage display, which takes advantage M13 bacteriophages to produce large peptide libraries. In this work, we aim to develop novel phage display techniques to improve its application toward the production of therapeutic peptides.

Herein we report two strategies to facilitate the incorporation of unnatural amino acids into phage-displayed peptide libraries and take advantage of these techniques to develop peptide inhibitors. A novel superinfection-immunity amber enrichment strategy was developed to afford the ability to perform phage-assisted active site directed ligand evolution (PADLE), where unnatural amino acids direct the enrichment of peptides that bind to a protein’s active site. By incorporation of lysine analogs into a phage-displayed peptide library, a peptide that inhibited Sirtuin 2 with low nanomolar potency was identified using in vitro studies. Following this, the development of a novel helper phage afforded an improved amber enrichment strategy for peptide libraries. Selections using these libraries were then performed against the extracellular domain of zinc and ring finger 3 (ZNRF3), a membrane bound E3 ubiquitin ligase. These selections not only identified ligands for ZNRF3, but also demonstrated the effects of noncanonical amino acids in phage selections.

Alongside the development of strategies for the incorporation of unnatural amino acids, we also report a novel organic linker, CAmCBT, to produce phage-displayed macrocyclic peptide libraries. Tuning the regioselectivity of CAmCBT for improved selectivity toward peptide libraries containing N-terminal cysteines, allowed for reduced toxicity in comparison with other linkers. CAmCBT was then used to generate a macrocyclic peptide library that was screened against epitopes of the SARS-CoV-2 spike protein. One of the enriched peptides, P29S1, exhibited low micromolar inhibition of SARS-CoV-2 infection through disrupting the Spike:ACE2 interaction.