Browsing by Subject "BPTF"

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    Development of peptide-based 19F MRI agents and BPTF-bromodomain inhibitors
    (2019-07) Kirberger, Steven
    Molecular imaging is the process of using targeted probes to detect abnormalities at the molecular level by observing interactions to specific biomarkers. Magnetic resonance imaging (MRI) presents an interesting avenue with respect to development of probes for the early detection of disease. In particular, 19F MRI shows promise for this development as the fluorine nucleus possesses many similar characteristics as the conventionally used proton but has the distinction of a lack of background signal found natively in biological systems. State of the art 19F MRI agents involve the use of perfluorinated compounds that often suffer from stability issues, bioaccumulation, as well as persistence within the environment. The first part of this dissertation describes the design and optimization of a peptide-based 19F MRI agent. These peptide scaffolds show promise for future use as 19F MRI probes due to their high signal, water solubility, and facile degradation in vivo to prevent bioaccumulation. The resultant byproducts have also been shown to be environmentally benign. This work is the focus of Chapters 2 and 3 of this document. In a second project, the development of a small molecule inhibitor of an epigenetic protein target is described. AU1, the first reported small molecule inhibitor of the bromodomain of a protein called BPTF, was discovered in the Pomerantz lab in 2015. As BPTF is a relatively understudied protein, there exists a need to improve the potency of AU1 as a probe for the various functions of its bromodomain, as it has been implicated in numerous diseases including: pancreatic cancer, melanoma, colorectal cancer, hepatocellular carcinoma, breast cancer, bladder cancer, and lung cancer. Structural analogs have been developed and analyzed in an attempt to improve upon AU1 in terms of its potency, solubility, and reduction of potential off-target binding. The work described in Chapter 4 of this document shows progress toward these goals, and the development of our 19F NMR assays for the analysis of protein ligands. A collaborative effort involving the (S)-enantiomer of AU1 is briefly described in Chapter 1. In a third section, the collaborative work between our lab and that of Ratmir Derda to develop peptide auxiliaries to improve therapeutic life-time in vivo is described. Chapter 5 details the use of 19F NMR to analyze the binding strength and location of numerous fluorinated peptides designed in the Derda lab.
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    Development of Protein-Observed Fluorine Nuclear Magnetic Resonance Spectroscopy as a Ligand Discovery Technique
    (2017-04) Urick, Andrew
    Fragment-based drug design (FBDD) has been rapidly gaining traction in the drug discovery process. A central tenant of fragment-based molecular screening is to use less sophisticated small molecules to sample chemical space more efficiently. With Vemurafenib and Venetoclax as FDA approved therapeutics from FBDD and several others in Phase III clinical trials, FBDD is becoming a validated technique for drug discovery. However, because of their small size these fragments are likely to bind to their target with a low affinity, necessitating more sensitive methods to detect protein-ligand interactions during a screen. Nuclear magnetic resonance spectroscopy has emerged as one of several powerful biophysical techniques for conducting fragment screens. In this thesis, a 19F protein-observed NMR method for detecting bromodomain−ligand interactions using fluorine-labeled aromatic amino acids due to the conservation of aromatic residues in the bromodomain binding site is described. Therein, we test the sensitivity, accuracy, and speed of this method with small molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment. Simplified 19F NMR spectra allowed for simultaneous testing of multiple bromodomains to assess selectivity and identification of a new BPTF ligand. Fluorine labeling only modestly affected the Brd4 structure and function assessed by isothermal titration calorimetry, circular dichroism, and X-ray crystallography. To benchmark its potential as a ligand discovery tool, we compare the protein-observed 19F NMR screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We conclude that for the protein class understudy here, protein-observed 19F NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. The speed, ease of interpretation, and low concentration of protein needed for binding experiments affords a new method to discover and characterize both native and new ligands.
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    Development of Small-Molecule Inhibitors and Biophysical Tools to Study the Epigenetic Protein BPTF
    (2022-06) Zahid, Huda
    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.