Ycas, Peter2022-03-172022-03-172020-01https://hdl.handle.net/11299/226665University of Minnesota Ph.D. dissertation.January 2020. Major: Chemistry. Advisor: William Pomerantz. 1 computer file (PDF); xviii, 296 pages.Epigenetics is the study of heritable changes in genome expression which alter organism phenotype. Genome expression is controlled by access to specific DNA, which in turn is controlled by how DNA associates with the histone proteins it is wrapped around. The condensed complex of DNA and histones is known as a chromosome. A hallmark of epigenetics is post-translational modification of chromosomes, both of DNA and histones. One such post-translational modification is acetylation of lysines on histone tails, a modification associated with gene transcription. The first part of this thesis focuses on bromodomain and plant homeodomain (PHD) finger transcription factor containing protein (BPTF), a member of the bromodomain class of proteins. Bromodomains recognize histone lysine acetylation and recruit transcription factors and nucleosome remodelers to chromatin. BPTF is a multi-domain protein which is the largest part of the nucleosome remodeling factor. The second half of this thesis examines another transcriptional regulator, the KIX domain of CBP/p300, a protein domain which co-localizes transcription factors. In Chapter one, the role of BPTF in gene expression and disease progression is discussed. The success of using small molecule probes to study other bromodomain containing proteins is described, highlighting the need to develop probes for BPTF in order to further discern the role of the protein in healthy and disease states. The current advances in small molecule inhibitor and biophysical tool development are described. The second half of Chapter one introduces the role of the KIX domain of CBP/p300 and its’ interaction partners and focuses on the past methods of studying the domain with a variety of NMR techniques. Chapter two describes the development and ligand deconstruction analysis of the first BPTF bromodomain inhibitor, AU1. The structure activity relationship of the molecule is developed using protein-observed fluorine NMR (PrOF NMR) and the (S) enantiomer of the compound is identified as the active component. Treatment of a panel of cancer cell lines which were found to be BPTF dependent through gene knockdowns showed decreased cell viability when treated with AU1. Development of biophysical tools to rigorously characterize high affinity BPTF inhibitors is described in Chapter three. The progress of ligand development in Chapter two was hampered by the lack of tools to accurately determine tight binding affinities. To address these issues, SPR and AlphaScreen assays were developed which are capable of rank ordering compounds which previously could not be done with PrOF NMR. These biophysical assays were validated against a small panel of previously characterized small molecule inhibitors of BPTF. Following validation, these assays were used to discover two new BPTF scaffolds which were explored as possible starting points for BPTF ligand discovery. Finally, the development of conditions to co-crystallize ligands with BPTF and the solution to their X-ray structure is described providing BPTF-small molecule ligand structural information for the first time. Chapter four compares fluorine labeling strategies of the KIX domain of CBP/p300 with 2-, and 3-fluorotyrosine (2FY and 3FY). The response of small molecule mimics of five fluorinated aromatic amino acids to changes in solvent polarity is used as a barometer for their utility in PrOF NMR. The chemical shift anisotropy of polycrystalline samples of 2FY and 3FY are determined by magic angle spinning. Through incorporation of both fluorinated tyrosine derivatives into the KIX domain of CBP/p300, their influence on stability and pKa perturbation in a model protein is investigated. The response of both 19F labeling strategies to ligand binding is discussed using the protein-protein interaction partner, MLL, which shows an allosteric response with 2FY KIX which is not observed with 3FY KIX. Chapter five delves into the differences observed with 2FY and 3FY KIX. The 19F NMR response of both proteins to a positive allosteric regulator (MLL), a negative allosteric regulator (naphthol AS-E phosphate), and an as yet unknown small molecule (2) are measured with changes in D2O solvent composition to measure the solvent accessibility of 19F nuclei upon ligand binding. The role of aromatic amino acids in the allosteric communication network is interrogated through ternary complex formation with MLL and c-Myb, which bind concurrently at separate sites on KIX. Finally, efforts towards crystallizing a stabilized complex of 2FY and 3FY KIX to discern their differences in allosteric communication are described.enBiophysicsChemical BiologyChemistryThesis or Dissertation