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Development of Phage Display Techniques with Genetic Code Expansion for Peptide Drug Discovery
Abstract
Phage display is one of the most widely-used techniques to develop peptide therapeutics. Its unique design links the displayed peptides to their encoding DNAs, providing a means for amplifying the peptide library and an easy way to identify selected ligands through sequencing. Although powerful, the conventional phage display technique replies on host cell’s translation machinery, therefore its chemical diversity is confined to twenty canonical amino acids. Further, phage-displayed peptides are also generally linear and unstructured leading to entropy penalty when binding to targets and proneness to proteolytic degradation.
To resolve these drawbacks, we expand the functional and structural diversity of phage display using orthogonal aminoacyl-tRNA synthetase/tRNA pairs to incorporate non-canonical amino acids with diverse chemical functionalities into displayed peptide libraries. First, we develop a system to genetically incorporate an N^ε-acryloyl-L-lysine (AcrK) at the C-terminus of an 8-mer library; the non-canonical amino acid undergoes a proximity-driven Michael addition with the cysteine at the N-terminus to generate a phage-displayed cyclic-peptide library. The cyclic peptide library is applied to the affinity selection against histone deacetylase 8 (HDAC8), leading to the discovery of a potent cyclic peptide inhibitor that binds to target protein with a single digit micromolar affinity and displays better potency than its linear counterparts. Then, the same approach is applied to screen against the spike protein of the novel coronavirus SARS-CoV-2 to evolve peptide inhibitors. A 12-mer library is constructed and used in a displacement-based selection which gives two peptide ligands; both are shown to disrupt Spike-RBD/ACE2 binding.
Lastly, a previously developed amber-obligate library is used to display an N^ε-butyryl-lysine (BuK) on 7-mer peptides. This lysine derivative is a naturally occurring lysine posttranslational modification that has target-ligand interactions with eleven-nineteen leukemia protein (ENL). During the selection, BuK serves as a warhead to guide displayed peptides towards the active site of ENL, thereby, increasing the selectivity and productivity of biopanning. We validate the selected peptides as ENL inhibitors, and further optimization and investigation have led to the discovery of a potent, cellular active peptide inhibitor that exhibits on-target effects in inhibiting ENL target gene expression and leukemia growth.
Citation
Chen, Peng-Hsun (2022). Development of Phage Display Techniques with Genetic Code Expansion for Peptide Drug Discovery. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /198683.