Browsing by Subject "Bromodomains"

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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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    Identification and Characterization of Potential BRDT(1) Inhibitors by Fragment-Based Screening Using Differential Screening Fluorimetry and Orthogonal Techniques
    (2019-05) Wisniewski, Andrea
    Almost 60 years after the advent of the female oral contraceptive pill, there are now a plethora of hormonal contraceptive options for women. Despite all of these available methods, 43% of pregnancies in the United states from 2011 to 2015 were unintended. Contraceptive methods for men are limited to condoms and vasectomies, neither of which offer complete protection from unintended pregnancies. Development of effective, reversible, nonhormonal male contraceptives would be a revolution in the area of contraception, complementing currently available methods and offering a new alternative to men. Epigenetics is a rising area of interest, concerned with the differential expression of genes caused by post-translational modification of proteins. Acetylation of lysine residues is regulated by several groups of proteins, including the bromodomains, responsible for recognizing the acetyl modifications and recruiting other effector molecules to impact chromatin remodeling, transcriptional control, and DNA repair. The bromodomain and extra terminal (BET) family of bromodomains has been implicated in many critical processes (such as cell cycle progression, transcriptional elongation) and disease states, including aggressive leukemias, obesity, acute inflammation, and NUT midline carcinoma. One BET protein, BRDT, is expressed exclusively in the testes and is responsible for regulating spermatogenesis. Knockout models of BRDT yield healthy, normal male offspring that are completely infertile. Studies using small molecule inhibitors of BET proteins have shown that BRDT inhibition causes a reversible infertility, making BRDT an attractive target for a non-hormonal male contraceptive. A fragment-based drug design strategy was employed to identify potential BRDT inhibitor leads. Differential scanning fluorimetry (DSF) was optimized for BRDT and used to screen a fragment library. An initial collection of 34 positive- and negative-shifting fragment hits were identified and confirmed from fresh powder by the DSF assay. Protein-observed 19F NMR (PrOF-NMR), fluorescence polarization (FP), amplified luminescent proximity homogenous assay screen (AlphaScreen), and x-ray crystallography were all part of the orthogonal screening cascade used to confirm BRDT binding. In total, 16 fragments were confirmed as ligands of BRDT, including a novel 1,5-benzodiazepinone scaffold. A number of these hits were pursued by our lab as lead molecules. In particular, and indanone scaffold was selected for further investigation. Analogs of the hit fragment were designed to develop structure-activity relationships of the chemotype; the indanone headgroup, linker composition, and substitution of a terminal phenyl group were studied. The analogs were tested by DSF, PrOF-NMR, FP, and AlphaScreen, with the most potent compounds submitted for crystallography experiments. The DSF assay developed for fragment screening was also used to test a number of other compounds for BRDT binding activity, including a pyridone scaffold identified by the DSF screen, several series based on kinase inhibitors shown to bind BET proteins, and a series based on a virtual high throughput screening hit.