The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery
Abstract
This study is composed of two parts. In the first part, we discussed nitrilimine-alkene cycloaddition for protein labeling. The mechanism of this nitrilimine-alkene cycloaddition was proposed, and thereby the best experimental condition for this protein labeling approach was investigated. The transient formation of nitrilimine in aqueous conditions is greatly influenced by pH and chloride. In basic conditions (pH 10) with no chloride, a diarylnitrilimine precursor readily ionizes to form diarylnitrilimine that reacts almost instantly with an acrylamide-containing protein and fluorescently labels it.
In the second part, a novel method for the synthesis of phage-displayed cyclic peptide libraries is presented. Cyclic peptide drugs are appealing in the drug discovery research area due to their unique advantages including high affinity, high specificity, low toxicity, and high cellular and serum stability. In order to identify cyclic peptides as therapeutic agents, during my graduate study I have developed a phage display-based methodology that integrates the genetic noncanonical amino acid (ncAA) mutagenesis technique for the synthesis of novel phage-displayed cyclic peptides through simultaneous 1,4-addition between a cysteine thiol group and acrylamide moiety in Nε -acryloyl-lysine (AcrK), a ncAA. Both cysteine and AcrK are genetically coded. The success of using a cysteine and an AcrK to cyclic a peptide in a model protein and phages was validated by various approaches. In order to afford a library, a phage-displayed cyclic peptide library was constructed by inserting a consecutive but sequence-randomized 6-mer peptide flanked by an amino side cysteine and a carboxyl side AcrK. Panning of the synthesized phage-displayed cyclic peptide library was performed against two target proteins that are tobacco etch virus (TEV) protease and histone deacetylase 8 (HDAC8). A lot of high-affinity phage clones were isolated and collected. DNA sequencing of these selected clones led to the identification of several peptides that potentially inhibit TEV protease and HDAC8. To confirm their potencies as inhibitors, abundant peptides and their fluorophore-conjugated derivatives were synthesized through solid-phase peptide synthesis (SPPS). Measurements of fluorescence polarization change and IC50 value of these peptides when they bound to TEV protease and HDAC8 were performed.
Overall, we have mechanistically characterized the nitrilimine-alkene cycloaddition reaction and developed a novel approach for the synthesis of phage-displayed cyclic peptide libraries. The selection of displayed peptides against TEV protease and HDAC8 has resulted in multiple peptides that display high potencies against these two enzymes.
Citation
Wang, Xiaoshan (2018). The Nitrilimine-Alkene Cycloaddition Mechanism and Phage-displayed Cyclic Peptide Libraries for Drug Discovery. Doctoral dissertation, Texas A & M University. Available electronically from https : / /hdl .handle .net /1969 .1 /173491.