Browsing by Subject "Bromodomain"

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    Computational Analysis on a Novel Set of 1,4,5 tri-Substituted Imidazole Based Bromodomain Inhibitors with Human BRD4
    (2020-05) Jones, Peter
    Bromodomain-Containing Protein 4 (BRD4) is a human transcriptional regulator and member of the N-Terminal Bromodomain and Extra Terminal Domain (BET) family of proteins. BRD4 binds to acetylated chromatin, preserving epigenetic modifications in the chromatin structure and activating the positive transcription elongation factor (p-TEFb) complex. This complex phosphorylates RNA polymerase II and promotes transcription of the immediate downstream genomic element. BRD4 shows promise as a target for anticancer therapies, with most research focusing on a class of drugs known as BET inhibitors. These drugs bind to the active site of BET family proteins, preventing BRD4 specifically from associating with chromatin. However, there is a lack of atomistic understanding regarding the binding of these drugs to BET family members. Many factors which influence the binding affinity of a series of 1,4,5 tri-substituted imidazole-based bromodomain inhibitors bound to the active site of BRD4 have yet to be characterized. Further, the effects of these inhibitors on the structural waters intrinsic to BRD4 remains unclear. Experimental work has suggested that the IC50 of this series of BET inhibitors could be explained in terms of a few specific interactions in the binding site. In this analysis, Free Energy Perturbations (FEP) are used to probe the relative free energy of binding for this set of differentially substituted BET inhibitors. Our working hypothesis is that the chlorine, bromine, and iodine substitutions participate in a halogen bond with the backbone oxygen of Met105 in BRD4, stabilizing the drug in the active site. Further, we propose that substitutions which cannot form this halogen bond, such as fluorine and other non-halogen substitutions, would have a higher free energy of binding. FEP analysis revealed that the chlorinated, brominated, and iodinated substitutions displayed a lower free energy of binding than the other substitutions, with evidence of a halogen bond between the drug and the backbone oxygen of Met105. It was also observed that this set of BET inhibitors displaces several highly coordinated solvent molecules in the active site of BRD4. By contrast, a simulation of BRD4 complexed with JQ1, another known BET inhibitor, does not displace these waters. These results support our hypothesis that a halogen bond is formed between the large halogen substitutions and the protein, increasing the binding affinity for substitutions that can participate in this type of interaction. This halogen bond can be exploited for improving this set inhibitors and designing novel compounds which bind more favorably to BRD4.
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    Fragment screening and biophysical method development for BET and non-BET bromodomain inhibitor discovery
    (2020-08) Johnson, Jorden
    Fragment-based drug discovery has become a popular and successful method for identifying small molecule modulators of various protein targets. Despite drugs and chemical probes having significant 3D-character, traditional fragment libraries are highly 2D with few sp3 centers, stereocenters, and unsaturated ring systems. The use of 3D-enriched fragments is highly debated especially regarding targeting protein-protein interactions. The two main concerns with using 3D-fragments are that the increased complexity of the compounds will cause prohibitively low hit rates and that 3D-compounds are not well suited for targeting the highly 2D protein-protein interaction interfaces. However, it is hypothesized that using 3D-fragments will allow for a fuller exploration of the binding pockets resulting in selectivity amongst targets at the onset of drug discovery. The first part of this dissertation details my work using 3D-enriched fragments for targeting bromodomains, a class of epigenetic proteins that function through protein-protein interactions but have a well-defined binding site. Mis-regulation of bromodomains has been associated with many disorders and cancers making them an attractive therapeutic target. However, gaining selectivity amongst the 61 bromodomain isoforms has proven challenging as they all recognize the same substrate, N-ε-acetylated lysine, a post translation modification on histones. In one project I used a 3D-enriched fragment library to target the first bromodomain of BRD4. In a second project, I developed a screening workflow for screening cocktails of fragments against two proteins simultaneously. Taking advantage of the pros and cons of ligand- and protein-observed NMR binding assays, I used protein- and ligand-observed NMR, I used protein-observed fluorine (PrOF) NMR and 1H CPMG NMR in concert, to screen a 3D-enriched fragment library against the bromodomains of BPTF and PfGCN5. In a third project, I worked to increase the efficiency of 19F labeling bromodomains for their use in PrOF NMR. Here I used tyrosine phenol lyase to make fluorotyrosine from fluorophenol in cellulo for direct incorporation into proteins sans isolation. Lastly, in several other project I worked to crystalize and solve seven bromodomain holo and cocrystal structures to aid in inhibitor development.
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    Part 1: Design and Synthesis of BRDT Selective Inhibitors as Male Contraceptive Agents Part 2: Focused Library Synthesis for TGR5 (Takeda G Protein-Coupled Receptor 5) Antagonist
    (2020-09) Jiang, Jiewei
    Unintended pregnancies can have significant adverse socioeconomic effects and health risks for women. One approach to reducing unintended pregnancies is the use of effective contraceptive methods. While women have multiple reversible contraceptive options, there is an unmet need for men to pursue safe and reversible infertility. Chapter 1 provides a brief overview of two pharmacological approaches to male contraception: disrupting the hormonal milieu (hormonal) and targeting key cellular components in sperm maturation and function (non-hormonal). Because of adverse effects resulting from hormonal disruption, we aim to develop safe novel non-hormonal male contraceptive agents. To this end, we have targeted the testis-specific bromodomain (BRDT), an epigenetic reader protein essential for spermatogenesis. Chapter 2 describes the validation of a tricyclic dihydropyridine hit from a virtual screening campaign. Based on the sequence alignment, we hypothesized that engaging the unique Arg54 in the first bromodomain of BRDT (BRDT-1), would achieve BRDT-1 selectivity over other bromodomain isoforms. Guided by this hypothesis, we explored three structural modifications on the scaffold: conversion its lactone functionality to a lactam, lactone ring-opening, and conformational restriction by macrocyclization. Cellularly active analogs with a greater than 10-fold increase in affinity were obtained. However, the desired BRDT-1 selectivity was not achieved for any of the three subsets. Additionally, novel mechanisms of action for targeting BRDT were pursued, including proteolysis-targeted chimeras (PROTACs) for selective protein degradation and bivalent molecules for simultaneous occupancies of two bromodomains. Chapter 3 focuses on an inherited genetic disorder, polycystic liver disease (PLD). The G protein-coupled receptor TGR5 activation was identified as strongly associated with PLD. To develop a TGR5 antagonist, we hypothesized that known TGR5 agonists could be converted to antagonists via systematic structural modifications. After selecting the nicotinamide core as our starting point, we used combinatorial chemistry to generate a focused library with over 100 analogs. The screenings for agonist and antagonist activity, however, only yielded TGR5 agonists rather than antagonists. Nevertheless, the results provide novel structure-activity relationship insight for TGR5 agonists based on the nicotinamide core. This experiment highlights the need to obtain additional information including the co-crystal structures for future TGR5 antagonist discovery efforts.