Browsing by Subject "Peptides"
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Item Development of peptide-based 19F MRI agents and BPTF-bromodomain inhibitors(2019-07) Kirberger, StevenMolecular 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.Item Nanomaterial solutions for the protection of insulin producing beta cells(2013-11) Atchison, Nicole Ann.Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) toβ cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat β cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.Item Synthesis and applications of caged thiols for studying protein prenylation.(2012-02) Abate Pella, DanielRas proteins are a subfamily of small GTP-binding proteins that are involved in various critical cellular processes including cell growth, survival and nuclear transport. It has been reported that roughly 30% of human cancers are derived from mutations of Ras, and prenylation is a key step that activates their oncogenicity. Commercial inhibitors of prenylation have been successful at arresting Ras activation and can be categorized into two families: farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase inhibitors (GGTIs). The focus of this thesis is to explore the use of photoremovable protecting groups (caging groups) to better understand the process of prenylation by caging the critical thiol residues of FTIs, GGTIs and peptides. The caging group bromohydroxy coumarin (bhc) was covalently bound to the thiol of the FTI L-744,832 in order to inactivate the inhibitor. This caged FTI was evaluated with respect to its one- and two-photon uncaging kinetics and ability to release FTI upon photolysis. Analysis shows that bhc photolysis occurs more rapidly compared to the most frequently used family of nitrobenzyl-based cages, and that FTI is produced with good yields upon one- and two-photon excitation. Bhc-FTI was then tested on different cell lines in order to show that upon irradiation FTI is released that inhibits Ras farnesylation (observed via Western blot analysis), Ras membrane localization (detected by confocal microscopy), and downstream signaling (fibroblast morphology). This same approach was utilized to cage FTI with bromohydroxy quinoline (BHQ). The covalent inactivation of FTI with BHQ was employed to cage the active site thiol (BHQ-FTI) and active site amine (BHQ-FTI urethane). Kinetic evaluation suggests that BHQ-FTI uncages faster than bhc-FTI but it produces little FTI upon photolysis due to the formation of unreactive photoproducts. Despite its poor yield, one photon cell experiments with BHQ-FTI resulted in the inhibition of Ras farnesylation, Ras membrane localization and downstream signaling. Quantitation and biological experiments with BHQ-FTI urethane are ongoing. Peptides that are substrates of protein farnesyltransferase (PFTase) were caged with bhc and BHQ at their crucial thiol that is targeted for farnesylation. Upon one-photon photolysis peptides caged with BHQ show poor yields of free peptide while bhc-caged ones result in good peptide production. One of these caged peptides was subjected to an in vitro farnesylation assay to show that no farnesylation occurs, but upon one- and two-photon irradiation farnesylated peptide can be detected. Application of this caged peptide to study the mechanism of farnesylation via X-ray crystallography is under way. Certain Ras proteins are alternatively geranylgeranylated and retain full function when farnesylation has been inhibited; as a result, GGTI-286 was caged with bhc to study this phenomenon. The synthesis of this GGTI and the inactivation of its thiol via covalent bonding with bhc is described here. The kinetic analysis of bhc-GGTI as well as its quantitation and biological testing are a work still in progress.