PROBING ALLOSTERY IN METHYL-LYSINE READER DOMAINS Abstract: Chromatin is the complex of histone proteins, RNA, and DNA that dynamically packages the genome within each eukaryotic cell. While cell lineage specific transcription factors clearly play a dominant role in the control of gene expression, the regulation of chromatin accessibility via post-translational modifications (PTM) of histones is of great current interest as the opportunities for pharmacological intervention in the action of the associated proteins are significantly better than in the direct perturbation of transcription factors by small molecules. The molecular details of chromatin regulation are just beginning to be understood and chemical biology is poised to play a central role in advancing scientific knowledge and assessing therapeutic opportunities in this field. Specifically, cell penetrant, high-quality chemical probes that modulate the regulation of chromatin state are of great significance. The advantages of a small molecule driven approach to exploring chromatin biology are numerous: temporal resolution; mechanistic flexibility; ease of delivery in cells and potentially, in vivo; and significantly, a chemical probe may provide an immediate transition to a drug discovery effort, possibly cutting years off the time between target selection and therapeutic intervention. This impact of a chemical probe results from simultaneously addressing target ‘validation risk’ (the likelihood that pharmacologic modulation of the target will have a favorable outcome in a disease) and ‘technical risk’ (the likelihood that a tolerable molecule that modulates the target can be discovered). While probes often lack some features required in drugs, their discovery diminishes many target validation and technical risks and creates a cascade of assays, structural and mechanistic information that is enabling to subsequent efforts focused on drugs. While high-quality probes are challenging to develop, they are achievable within the resources available to academic programs, and their creation is a fantastic training experience. To maximize the impact of our probes, we intend to continue our approach of sharing them without creation of intellectual property. We have pioneered a target-class probe discovery strategy within the large family of methyl-lysine (Kme) readers. We have been productive in this area and built momentum for future studies focused on the allosteric interactions between Kme reader domains, nucleotide binding domains, and the catalytic domains that regulate chromatin function. During this effort, we have established a network of talented collaborators that complement our strengths in chemical biology, medicinal chemistry, in vitro assay development, and biophysics; with strengths in molecular, structural and chromatin biology. Allosteric interactions in chromatin regulatory complexes are critically important phenomena that create unique opportunities for pharmacologic interventi...