An amber obligate active site-directed ligand evolution technique for phage display

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.

Chemical Diversification of Simple Synthetic Antibodies

“…52,123 In this regard, the simple synthetic antibodies and characterization strategies elucidated in this work could facilitate sideby-side evaluations of multiple chemical modification strategies. Finally, while many routes to prepare chemically modified peptides in display formats are now available, 29,[35][36][124][125][126][127][128][129][130][131] very few analogous approaches for the chemical diversification of proteins have been described. 53,60 The efficient preparation and chemical diversification of antibodies on the yeast surface opens up new possibilities for discovering "drug-like" protein leads in high throughput.…”

Mechanism-based inhibitors of SIRT2: structure–activity relationship, X-ray structures, target engagement, regulation of α-tubulin acetylation and inhibition of breast cancer cell migration

“…This further enabled us to glean insight into the binding kinetics of SIRT2 inhibition by applying continuous assay formats, which has not been achieved with SIRT2 deacetylation assays. 102 Standard end-point dose-response assays do not take into account the kinetic behavior of inhibitors, 80,81 which may give rise to IC 50 values that vary substantially with specific assay conditions, especially if the inhibitor does not exhibit standard fast-on/fast-off kinetics. 103 It is well documented that mechanismbased thioamide-and thiourea-containing inhibitors may form stalled intermediates with ADPR in the enzyme active sites, which can affect binding kinetics.…”

Mechanism-based inhibitors of SIRT2: structure–activity relationship, X-ray structures, target engagement, regulation of α-tubulin acetylation and inhibition of breast cancer cell migration

“…This further enabled us to glean insight into the binding kinetics of SIRT2 inhibition by applying continuous assay formats, which has not been achieved with SIRT2 deacetylation assays. 102 Standard end-point dose-response assays do not take into account the kinetic behavior of inhibitors, 80,81 which may give rise to IC50 values that vary substantially with specific assay conditions, especially if the inhibitor does not exhibit standard fast-on/fast-off kinetics. 103 It is well documented that mechanism-based thioamide-and thiourea-containing inhibitors may form stalled intermediates with ADPR in the enzyme active sites, which can affect binding kinetics.…”