Developing small molecule ligands for the study of bromodomain-histone interactions

Dynamic changes in DNA methylation, and post-translational modifications on histone proteins which DNA wraps around, form the basis of epigenetic transfer of information. The histone code refers to different combinations of epigenetic marks, each mediating transcription in a specific manner. Bromodomains are acetyl-lysine readers of the histone protein which interact selectively with acetylated lysine residues on histones. This protein-protein interaction alters gene expression levels, causing specific downstream effects. The human bromodomain family consists of 61 unique proteins, which are divided into eight subfamilies based on sequence similarity. Most research has been focused on the bromodomain and extra C-terminal (BET) family, which has yielded 14 compounds currently in clinical trials. However, only a few selective ligands and no compounds in clinical trials have been reported for other bromodomain families. Herein is reported, an extensive structure activity relationship of a potent CREBBP bromodomain ligand. The focus was on the cation-π interaction between R1173 and the tetrahydroquinaxolinone, and a good correlation between the computationally calculated electrostatic surface potential and the CREBBP bromodomain binding affinity was observed. Attempts to increase the selectivity of the ligand over the BET bromodomain were unsuccessful. The stability of the dihydroquinaxolinone headgroup was optimised by ring size expansion, which maintained CREBBP affinity and binding mode â the latter of which was confirmed by X-Ray crystallography. The propyl linker was modified by introducing a gem-difluoro group, and due to the conformational effect of the gauche-gauche interaction, a 3-fold affinity increase was observed. A second project in this dissertation reports the development of a small, modular photo affinity based probe based on a 3,5-dimethylisoxazole acetyl lysine mimic. This probe could be crosslinked to purified bromodomain proteins with up to 30% efficiency and subsequently modified via CuAAc chemistry to attach a fluorophore or biotin tag. The probe can crosslink to BRD4(1), CREBBP, WDR9(2) and the parasitic bromodomain TcBDF3.