http://hdl.handle.net/10027/19 UIC College of Pharmacy Fri, 24 May 2013 18:23:41 GMT 2013-05-24T18:23:41Z http://hdl.handle.net/10027/9803 Exploration of Histone Deacetylase Ligand Binding Modes by Photoaffinity Probes Histone deacetylases (HDACs) regulate chromatin structure and function. Since much of the literature has reported aberrant expression and recruitment of HDAC in malignant tissues, the role of HDACs in regulating the genes that are involved in cell cycle progression and arrest makes them attractive therapeutic targets for the treatment of cancer. Even though the pan-HDAC inhibitor showed promising antiproliferative activity, their application may be limited due to their toxicity and negative side-effects. To address this gap, design of isoform-selective HDAC inhibitors might be of significant importance for the treatment of cancer, making the knowledge of how inhibitors interact with HDACs and their binding mode crucial for isoform-specific inhibitor discovery. This study focused on investigating the interaction between HDAC probes and HDACs at the molecular level by using a combination of in vitro bioassays, immunoblotting, fluorescence spectroscopy, and protein mass spectrometry. In this dissertation, a binding ensemble profiling with (f)photoaffinity labeling approach that utilizes photolabeling of HDAC8 or HDAC3/NCoR2-DAD with a probe containing a UV-reactive aromatic azide, mapping of the covalent modification by liquid chromatography-tandem mass spectrometry, and a computational method to characterize the binding poses of the probe is described. 1) Two distinct binding poses of the HDAC8 probe were identified. An “upside-down” pose with the surface binding group of the probe bound in an alternative pocket near the catalytic site may contribute to the binding. 2) Modification sites on HDAC3 and NCoR2-DAD were identified and the interaction between HDAC3 and its corepressor NCoR2-DAD was explored. The results presented in this study showed that our combined approach could not only delineate the regions involved in the interface between two interacting proteins but also lead to structural models where the relative orientation of the proteins and their accessibility by small molecule inhibitors are defined. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9803 2013-02-21T06:00:00Z http://hdl.handle.net/10027/9795 A Novel Anti-Diabetic Peptide Nanomedicine Against Pancreatogenic Diabetes A Novel Anti-Diabetic Peptide Nanomedicine Against Pancreatogenic Diabetes Amrita Banerjee, Ph.D. Department of Biopharmaceutical Sciences University of Illinois at Chicago Chicago, Illinois (2012) Dissertation Chairperson: Hayat Onyuksel, Ph.D. Pancreatogenic diabetes (PD) is a potentially fatal disease that occur secondary to pancreatic disorders such as chronic pancreatitits. The current anti-diabetic therapy for PD is fraught with many adverse effects that can increase morbidity. This research is geared towards the development of a novel peptide nanomedicine: pancreatic polypeptide (PP) in sterically stabilized micelles (SSM) for management of PD. PP is a hormone secreted by the pancreas mostly post-prandially and has well documented anti-diabetic efficacy. The peptide is known to reverse hepatic insulin sensitivity and improve glucose tolerance. However, the short plasma half-life of PP limits its therapeutic application. In order to prolong and improve the activity of PP in vivo, we proposed to deliver the peptide in SSM. The hypothesis of our research was that PP would self-associate with SSM to form a novel peptide nanomedicine and demonstrate effective anti-diabetic activity against pancreatogenic diabetes. We have successfully prepared the formulation of PP in SSM (PP-SSM) and characterized it in terms of its saturation molar ratio of association with phospholipid micelles, particle size distribution, conformation as well as stability of the formulation towards both freeze-drying and against proteolytic degradation. We observed that presence or absence of sodium chloride in aqueous media dictated the association of PP with SSM, the particle size distribution and the stability of the formulation towards lyophilization. PP retained its native conformation in SSM and the micelle associated peptide was more stable to proteolytic degradation than free peptide in buffer. Through in vitro cell studies we found that PP in SSM exhibited similar bioactivity as peptide in buffer. Results from in vivo experiments involving administration of PP-SSM to rodent model of PD indicated that PP-SSM significantly improved glucose tolerance, hepatic insulin sensitivity and increased hepatic glycogen content as compared to PP in buffer. The results of the study established the importance of micellar nanocarriers in protecting enzyme-labile peptides in vivo and delivering them to the target site, thereby enhancing therapeutic efficacy of these biomolecules. In summary, PP-SSM demonstrated promising anti-diabetic efficacy and therefore the nanomedicine should be further developed for effective management of pancreatogenic diabetes in the clinics. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9795 2013-02-21T06:00:00Z http://hdl.handle.net/10027/9783 Multivalent Binding, Cell Rolling, and Micropatterning for Enhanced Detection of Circulating Tumor Cells Circulating tumor cells (CTCs) in the blood of cancer patients are related to cancer progress and metastasis. CTCs can be isolated from non-epithelial, hematological cells using one of antibodies against epithelial cell surface markers called epithelial-cell-adhesion-molecule (EpCAM), human-epidermal-growth-factor- receptor-2 (HER-2), and prostate-specific-antigen (PSA). However, the effective detection of CTCs has a challenge coming from the extreme rarity of CTCs (approximately one tumor cell in the background of one million - one billion blood cells). Recently, we have developed a novel surface system that is programmed to mimic two naturally occurring processes to detect these rare cells at great sensitivity and selectivity: i) E-selectin- mediated cancer cell rolling and ii) the multivalent binding through multiple antibodies immobilized on a dendrimer. A poly(amidoamine) dendrimer, a spherical polymer with multiple reactive groups on its surface, was used to mediate the multivalent binding effect. The biomimetic combination of E-selectin and antibody- dendrimer conjugate was micropatterned on a multifunctional surface using a gasket for effective recruitment and specific isolation of tumor cells, respectively. With in vitro spiked cancer cells in culture media or human blood, the multifunctional surfaces resulted in a significantly enhanced tumor cell detection by ~39-fold at maximum with higher binding stability than the control surfaces. This study demonstrates a novel surface engineering approach to exploiting the E-selectin-mediated cell rolling and strong multivalent binding, which has great potential for clinically significant detection of CTCs. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9783 2013-02-21T06:00:00Z http://hdl.handle.net/10027/9781 Pharmacognosy of Raw Materials for Black Cohosh Dietary Supplements This study explored the potential of the aerial parts of Cimicifuga racemosa as a sustainable source of raw materials for the production of black cohosh dietary supplements to promote women’s health. The first step in this process was to isolate and identify characteristic secondary metabolites from the aerial parts of C. racemosa and compare these metabolites with those known from the roots which are believed to have therapeutic effects. The composition of extracts of the aerial parts of C. racemosa was shown to be different compared to the extracts of the roots/rhizomes. Two new cycloartane triterpene glycosides, 24-epi-1alpha-hydroxycimigenol-3-O-beta-D-xylopyranoside and 1alpha-hydroxydahurinol-3-O-beta-L-arabinopyranoside, were isolated and their structures elucidated. In addition to the two new compounds, five triterpenes were isolated for the first time from C. racemosa. The next step was to establish a method for differentiation and identification of Cimicifuga species and plant parts in black cohosh dietary supplements. A statistical method using Principal Component Analysis (PCA) of proton NMR spectra was employed to take into account small signals in the NMR spectra for the identification of extractives from the Cimicifuga species. PCA scores enabled the identification of both the aerial parts and the roots/rhizomes of each of the three of American Cimicifuga species in crude extracts. This metabolomic approach complements the first goal, aimed at the profiling of secondary metabolites of the roots/rhizomes, by taking into account unknown/unidentified compounds. The third step was to evaluate the biological potential of Cimicifuga racemosa aerial parts extract and compounds in terms of safety and efficacy parameters, including chemoprevention, estrogenicity, serotonergic activity, and drug interactions. In conclusion, it was determined that aerial C. racemosa does have potential to be developed into a botanical dietary supplement that may be able to substitute black cohosh products that are currently produced from wild-crafted below-ground parts. Thu, 21 Feb 2013 06:00:00 GMT http://hdl.handle.net/10027/9781 2013-02-21T06:00:00Z