Development of Small-Molecule Inhibitors and Biophysical Tools to Study the Epigenetic Protein BPTF

Abstract

Bromodomains are protein-protein interaction modules involved in epigenetic regulation of gene expression, typically through the recognition of acetylated lysine residues in histones. Bromodomain-containing proteins are involved in several disease processes, including cancer, inflammation and viral replication. There are 61 known human bromodomains and due to their high structural similarity, developing small-molecule inhibitors that bind with high affinity to specific proteins is a major challenge in the field. While numerous small molecule probes have been developed for the BET (bromodomain and extra terminal) family, few have been reported for the 53 non-BET bromodomains. Potent and selective chemical probes are, therefore, required to enhance our biological understanding of the non-BET bromodomain family. Among them, the understudied protein Bromodomain and PHD finger Transcription Factor (BPTF) is known to play an important role in chromatin remodeling and is the focus of this dissertation. BPTF is overexpressed in several cancers such as melanoma, breast cancer and high-grade gliomas. It has been identified as a potential target for developing cancer therapeutics and BPTF bromodomain inhibitors are being explored for combination treatment with chemotherapeutics. Given the emerging significance of BPTF as an anticancer target, small-molecule inhibitors for this bromodomain-containing protein are needed. This dissertation details efforts to develop potent chemical inhibitors of the BPTF bromodomain through systematic structure-activity relationship analyses. Starting from a reported pyridazinone scaffold and using structure-based design approaches, the lead compound BZ1 is developed with a Kd of 6.3 nM for BPTF, making it one of the most potent inhibitors reported to date for this bromodomain. These efforts are assisted by the optimization of a bead-based competition assay AlphaScreen (described in Chapter 2) which is used to quantify affinity values and cross-validate other biophysical methods for studying BPTF interactions. Several of the first cocrystal structures of BPTF bound to small-molecule ligands are also reported, which are key to rational design of potential drug candidates. We further show that these compounds sensitize breast cancer cells to the chemotherapeutic doxorubicin, indicating that inhibition of the BPTF bromodomain can overcome chemoresistance in these cells. Efforts are now underway to evaluate the drug-like properties of pyridazinone inhibitors for in vivo studies. The development of potent BPTF inhibitors paved the way to heterobifunctional molecules described in Chapter 4 of this dissertation. In this study, degraders based on a pyridazinone scaffold are designed and evaluated using an in-cell NanoBRET assay. As first-generation degraders, these compounds induce ternary complex formation of the BPTF bromodomain with the cereblon E3 ligase, evident through both in vitro and in-cell assay formats. Using an optimized NanoBRET assay, we show that they also efficiently degrade a Nanoluciferase-BPTF bromodomain construct. While preliminary western blotting studies indicate that these pyridazinone-based degraders do not show any degradation activity for endogenous BPTF, future work will look at other cell lines and various linkers to optimize the degradation efficiency of these compounds. In another project, detailed in Chapter 5, a fundamental study of noncovalent and highly stabilizing sulfoxide-aromatic interactions in proteins is described. This will provide insight into the effects of oxidation of methionine residues in α-synuclein, a modification known to be involved in neurodegenerative disorders such as Parkinson’s disease. β-hairpin peptides are used as model systems to study these motifs and quantify the interaction of oxidized methionine with aromatic side chains of amino acids. In this system, no additional stabilization is observed when interactions of phenylalanine with methionine and oxidized methionine are compared. Further analysis with tryptophan indicates a slight destabilization of the β-hairpin peptide motif with oxomethionine. Follow-up studies for this project will look at other aromatic side chains such as tyrosine and the application of this approach in the broader context of α-synuclein.