Chemical probe development for bromodomain and PHD finger-containing transcription factor (BPTF) reader domains as anti-cancer therapy

Abstract

Epigenetics is the study of mechanisms that result in differential gene expression without changes to the underlying DNA. In cancer, there is a dysregulation in gene expression, which can be driven by epigenetic proteins. One such protein implicated in numerous cancers is BPTF (bromodomain and PHD finger-containing transcription factor), an epigenetic protein whose main function is the recognition of post-translationally modified histones within chromatin and is the largest subunit of the nucleosome remodeling factor complex. BPTF binds chromatin via interactions with acetylated histone H4, (H4K16ac), through a structural motif called a bromodomain and trimethylated histone H3, (H3K4me3), via its C-terminal PHD finger to facilitate chromatin remodeling. BPTF’s oncogenic function is in part associated with increased chromatin accessibility and a direct protein-protein interaction with the oncoprotein c-MYC. Together, this makes BPTF a promising anti-cancer drug target. Therefore, the design of chemical probes for both reader domains (bromodomain and PHD finger) would be advantageous to better understand disease function.The first part of this thesis will detail structure-based design of BPTF bromodomain inhibitors. In Chapter 2, I describe the development of BZ1, the most potent reported BPTF bromodomain inhibitor (Kd = 6.3 nM) at the time. Design was aided by SAR around a pyridazinone scaffold and co-crystal structures, where we identified an acidic triad as a targetable feature for potency and selectivity gains in future inhibitor development. We were able to show that these tool compounds synergistically sensitized chemotherapeutics in a triple negative breast cancer cell line via on-target activity. While BZ1 has >350-fold selectivity over BET bromodomains, top-off targets included the bromodomains of BRD9 and CECR2. In Chapter 3, I describe efforts targeting a structured water in the BPTF binding site to improve selectivity. I optimized competitive assays for rapid off-target quantification, that led to second generation inhibitor, BZ2, which has improved selectivity over BRD9 and CECR2. Furthermore, we began to use the BZ scaffold to identify cell lines sensitive to BPTF inhibition and develop heterobifunctional molecules. The second part of this thesis will describe a screening effort that led to the discovery of the first reported chemical matter for the BPTF PHD finger. In Chapter 4, I describe a fragment-based screen using 1H CPMG NMR, which had a 5.7% hit rate against BPTF PHD. Eight hits were validated in a secondary direct binding assay, and further identified to compete with the histone peptide. Two scaffolds, pyrrolidine- and pyridazine-containing fragments respectively, were prioritized by SAR by catalog. Molecular docking and HSQC NMR began to elucidate binding interactions. In Chapter 5, I continued developing assays to investigate ligand-protein binding interactions, as well as preliminary selectivity tests against a related PHD finger, KDM5A PHD3. Additionally, SAR around one of the hit scaffolds revealed potential directions for future inhibitor design. In the future, these approaches will allow for understanding of the PHD fingers biological role in disease, as well as dual domain inhibition. Finally, Appendices A-B describe support to collaborative projects in a structural biology capacity. For our BRD4 project, another bromodomain we study in the Pomerantz lab, I solved a co-ligand crystal structure to aid in BRD4 inhibitor design (PDB: 7MLS). For an alpha synuclein project in the Sachs lab that was investigating the effects of pH in 1H,15N-HSQC NMR spectroscopy, I assigned resonances and generated chemical shift plots to determine what resonances were significantly perturbed by pH.