Browsing by Subject "Fluorine"
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Item Applications of Fluorine Magnetic Resonance for Small-Molecule Screening, Ligand Development, and Oxygen Sensing(2017-08) Gee, CliffordProtein-protein interactions (PPIs) play a vital role in biological processes but are difficult to target therapeutically. However, targeting PPIs is an important challenge because their dysregulation is linked to many various disease states including cancers and neurological disorders. While high throughput screening (HTS) has long been the standard method for drug discovery, fragment-based screening (FBS) has emerged as a promising alternative strategy due to its greater coverage of chemical space with smaller library sizes. Successful cases like Vemurafenib and Venetoclax, continue to bolster FBS efforts. Though many techniques, including X-ray crystallography, surface plasmon resonance, and thermal shift assays, have all been used as screening tools, the central hypothesis of this dissertation is that 19F NMR is a powerful and time efficient FBS tool that is complementary to existing tools and is useful for characterizing proteins and small molecule ligands. Protein-Observed Fluorine NMR Spectroscopy (PrOF NMR) due to its high speed, lack of background signals, environmental sensitivity, is an ideal method to use for both ligand discovery and characterization of ligand-protein interactions. Herein, we describe the application of PrOF NMR to two proteins in particular, the KIX domain of CBP/p300 which is part of a larger transcriptional activation complex, and the first bromodomain of BrdT, an epigenetic “reader” protein that has been validated as a target for male contraception. We demonstrate the use of PrOF NMR as a primary screening tool for KIX, identifying key pharmacophores for KIX binding. We also demonstrate the use of PrOF NMR for characterizing ligand-protein interactions, uncovering a new binding site in KIX, distinct from its two native transcription factor binding sites. Validation of hits from other screening campaigns can also be followed via PrOF NMR, and the quantitative information obtained can be used to guide the structure-activity relationship (SAR) process for further ligand development. Beyond ligand discovery in proteins, fluorine magnetic resonance can also be applied as an imaging and oximetry tool. Given the sensitivity of fluorine and its applications in both biophysical and biomedical contexts, fluorine magnetic resonance serves as a new tool for small-molecule screening, ligand development, and oxygen sensing.Item Development of Protein-Observed Fluorine Nuclear Magnetic Resonance Spectroscopy as a Ligand Discovery Technique(2017-04) Urick, AndrewFragment-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.Item Fluorinated Metal Complexes as MRI Contrast Agents(2015-08) Weitz, EvanMagnetic resonance imaging (MRI) is a vital tool in today’s modern healthcare system. MRI is preferred over positron emission tomography (PET) and X-ray computed tomography (CT) because it is non-invasive, non-radioactive, and provides 3-D imaging directly in vivo. Contrast agents are used in order to enhance the resolution of the images from MRI. All currently used contrast agents are based on gadolinium and image water protons in the human body. However, gadolinium-based contrast agents are principally unable to quantitatively image specific biomarkers of diseased states, lacking a ratiometric mechanism. Fluorine-based MRI does not suffer from these limitations, but its low sensitivity, with a limit of detection (LOD) in the micromolar range first requires a contrast agent designed specifically to address this issue of sensitivity, which can be accomplished using contrast agents with an incorporated lanthanide center. Fluorine MRI eliminates background signals and has a large chemical shift range which enables fluorines in different environments to each be imaged independently. This in turn allows for the development of ratiometric, responsive contrast agents whereby the total probe concentration and the concentration of the analyte can be independently determined. In this thesis, the theory, practicality, utility, and potential for fluorine-based MRI contrast agents is described. Sensitivity is addressed, synthesis is described, and demonstrations of the potential for fluorine MRI are examined in vitro and in vivo in order to design highly-sensitive, responsive, and biocompatible fluorine contrast agents.