Abstract
Although noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. Here, we report a technique based on superinfection immunity of phages to enrich amber-containing clones, thus avoiding the observed bias that has hindered incorporation of ncAAs into phage libraries. We then take advantage of this technique for development of active site-directed ligand evolution of peptides, where the ncAA serves as an anchor to direct the binding of its peptides to the target’s active site. To demonstrate this, phage-displayed peptide libraries are developed that contain a genetically encoded butyryl lysine and are subsequently used to select for ligands that bind SIRT2. These ligands are then modified to develop low nanomolar inhibitors of SIRT2.
Highlights
Noncanonical amino acids were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages
Inspired by the phage display technique that allows for easy construction of a large peptide library (>109) and creates a physical link between displayed peptides and their encoding DNA for efficient enrichment and identification of peptides that are bound to a particular target, we sought to combine a part of the structure–activity relationship (SAR) concept with the phage display technique to develop an amber-obligate phage-assisted, active site-directed ligand evolution technique for the rapid identification of potent ligands for epigenetic regulators
In order to carry out our proposed phage-assisted, active site-directed ligand evolution, it is necessary that all clones in a phage display library express peptides that contain a ligand-fused noncanonical amino acids (ncAAs)
Summary
Noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. A number of post-translationally modified lysines and their analogs have been genetically incorporated using the amber suppression method (Fig. 1c)[15,16,17,18,19,20,21,22,23] These ncAAs can serve as binding anchors for a number of epigenetic regulators to quickly evolve their potent and selective ligands using our proposed technique. Substitution of thiobutyryl (tBuK) and thiomyristoyl (tMyK) analogs into the selected peptides results in inhibitors of SIRT2 that are selective against other sirtuin isoforms and much more potent than previously reported small-molecule inhibitors, TB and TM Given these results and current abilities to genetically encode post-translational modifications, this technique is useful in identifying inhibitors of various epigenetic readers, writers, and erasers
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
