The long term goal in this research is to inhibit the function of epigenetic effector or ?reader? domains within a bromodomain and-PHD-finger-containing transcription factor, BPTF. We hypothesize BPTF, via its chromatin binding and remodeling function, controls an essential regulatory network that supports cell proliferation and cell cycle progression in cancer. Our preliminary data using BPTF genetic knockdowns and small molecule inhibitors supports a tumorigenic role of BPTF in breast cancer, consistent with findings in melanoma, bladder, lung, and pancreatic cancer. The inhibition of the oncogenic function of BPTF is highly significant, and offers a new molecular target for cancer therapy. The goals of this research are to determine the the role of bromodomain function by developing small molecule inhibitors and testing their activity using biophysical, biochemical, and cell-based models. Using shRNA and lead molecule S-AU1, we have established transcriptional reporter, cellular proliferation, and RNAseq assays in breast cancer cell lines as new tools to study the role of our molecules in a cellular setting. To complement traditional genetic approaches, a ?chemical knockout? tool will also be used for recruiting a ubiquitin ligase to BPTF using bifunctional molecules containing a bromodomain-targeting group and an E3-ubiquitin ligase ligand for inducing proteosomal degradation. The innovation behind our approach uses a new multi-protein 19F NMR protein-based methodology for the discovery of isoform selective bromodomain inhibitors. Due to the hyper-responsive nature of fluorine to ligand binding, the result is a rapid 1D NMR experiment, that is well-suited for structure-based small molecule studies. Due to the simplicity of the NMR spectrum, highly homologous BPTF and Brd4 bromodomains will be tested together to readily assess selectivity of optimized molecules. This method has already led to the discovery of the first small molecule for the BPTF bromodomain, AU1. This molecule will be further optimized in addition to molecules from a parallel ligand discovery platform using small molecule microarrays in Aims 1 and 2. In aim 3, a new bifunctional molecule based on AU1 is proposed for recruiting enzymatic functionality of ubiquitin ligases for selective proteolytic degradation of BPTF. This proposal has broad biomedical significance as a general method for early lead discovery and studies of epigenetic reader domains important in human health and disease. Although the role of the BPTF protein in cancer has been established, the first potent chemical probes for BPTF reader domains have potential for high impact as new chemical tools for inhibiting cancer progression. To address this goal, we have established a team of experts in their respective fields of chemical biology, cancer biology, and structural biology. Given that transcription factors represent a major class of potential drug targets, our new 19F NMR small molecule discovery method and cell-based approaches for epigenetic reader domains described here could significantly increase the repertoire of targets and thereby open up new avenues for drug discovery.