Methyl reader proteins recognize and bind to post-translationally methylated residues and have functional roles in diverse cellular processes including gene regulation, development and oncogenesis. The CBX polycomb paralog family of methyllysine readers recognize trimethyllysine lysine residues on histone tail 3 and repress transcription by compacting chromatin. The polycomb paralogs form multi-protein complexes that silence the expression of tumour suppressor genes, and play important roles in regulating cell cycle and differentiation. Each paralog is structurally similar, yet has distinct functions, of which many are unknown. My work has focused on the design and synthesis of CBX inhibitors and on the development of new methodologies for the discovery of inhibitors targeting methyllysine readers. In this work, I report on a series of potent peptidic inhibitors that selectively target the CBX polycombs, as well as the first selective inhibitor for the family member CBX6, and dual-active inhibitors that target CBX6/CBX8. The results demonstrate the potential to achieve selectivity through interactions outside of the methyllysine binding domain. Structural determinants in the binding pocket of each protein that differ within the family and give rise to selectivity were discovered. I will also report on a series of peptidomimetic CBX inhibitors that are active in cells. Cellular active inhibitors are critical for understanding the biological role of each CBX protein and their potential as therapeutic targets. New high-throughput approaches are needed to efficiently target methyllysine readers by chemical inhibition. I describe in this work a strategy for creating massive libraries of phage-displayed peptidic inhibitors containing methyllysine mimics. Synthetic optimization on cysteine-containing peptide phage constructs allowed for the successful installation of Kme3 mimics. This is the first report of a post-translational methylated peptide phage library. The methodology I developed can be used in a synthetic chemistry-driven adaptation of traditional phage display for the screening of millions of peptide-based compounds. Strategies that allow for diversity and high throughput screening will aid in future efforts in targeting the highly similar CBX proteins. / Graduate / 2021-06-01