http://hdl.handle.net/2104/4476 2013-05-23T21:02:09Z http://hdl.handle.net/2104/8597 High-throughput analysis of emerging and historical pollutants in biological matrices. Subedi, Bikram. The Deepwater Horizon Gulf Spill highlighted key deficiencies associated with our ability to analyze contaminants in biological tissue in a timely manner. These deficiencies in laboratory capacity and preparedness demonstrated a critical need to develop and validate high-throughput analytical methods capable of rapid and accurate quantification of organic pollutants in biological tissue from aquatic environments. High-throughput methods offer distinct advantages over historical methods, namely a reduction in the intrinsic costs associated with the sample preparation: time, solvents, labor, laboratory space, training, and potential loss of analytes. Sample preparation steps for the analysis of organic pollutants represent ~2/3 of the analytical cost, and are often the logistical bottleneck for laboratory capacity and preparedness. An enhanced pressurized liquid extraction (ePLE) method was developed for simultaneous analysis of pharmaceuticals and personal care products (PPCPs) in fish tissue and was utilized in a nationwide PPCP study conducted in Germany. The developed high-throughput method incorporated silica gel cleanup with PLE and improved the method sensitivity by optimizing resonant collision induced dissociation energy through ion-trap mass analyzer. The nationwide PPCP study found that the proximity of sampling locations to the upstream wastewater treatment plant discharging point and mean annual flow at the sampling locations were found to significantly powerful predictor of galaxolide and tonalide fish tissue concentrations (galaxolide: r2 = 0.79, p = 0.021 and tonalide: r2 = 0.81, p = 0.037) in Germany. Galaxolide and tonalide, both polycyclic musk fragrance materials, in German fish tissues were ~19 and ~28× lower, respectively, as compared to similar nationwide PPCP study in the United States. Similarly, a high-throughput ePLE method was developed for polychlorodibenzo-p-dioxins/furans (PCDD/Fs) and dioxin-like polychlorobiphenyls (dl-PCBs) by incorporating pressurized liquid extraction and multiple cleanup techniques. Sample preparation time and solvents were reduced as much as 95% and 65%, respectively, as compared to a conventional USEPA Method 1613. ePLE extracts were sufficiently clean for high resolution mass spectrometry analysis of PCDD/Fs and dl-PCBs in fish tissues from San Jacinto River Waste Pits, a Superfund site in Houston, TX. 2013-05-15T00:00:00Z http://hdl.handle.net/2104/8568 Kinetic characterization of thiosemicarbazones as cysteine protease inhibitors and their potential use as therapeutic agents against metastatic cancer and Chagas' disease. Chavarria Nolasco, Gustavo E. Human cathepsins L and K are cysteine proteases that play important roles in physiological and pathological processes, such as cancer metastasis, bone resorption and neurodegenerative diseases. This research has focused on the evaluation of synthetic thiosemicarbazones that could inhibit their proteolytic activities with the objective of preventing cancer metastasis. In addition, cruzain, a cathepsin L-like cysteine protease found in Trypanosoma cruzi, is a validated target for pivotal roles in the parasitic invasion in Chagas' disease, a condition that could be fatal if not treated. Currently, there is no effective treatment against the disease, which is rapidly extending to non-endemic areas in the United States and Europe. More than 150 synthetic thiosemicarbazones (obtained through a collaborative study) were tested against cathepsins L, K and cruzain. This work presents preliminary in vitro analysis of these compounds in order to characterize their potency and mode of inhibition. A number of potent inhibitors was found for each enzyme. A smaller subset of thiosemicarbazones were found to be selective. Results showed that compound 1, one of the most potent inhibitors in this library is a slow binding, slowly reversible, competitive inhibitor of these targets. Furthermore, 1 was able to delay and partially inhibit the activation of procathepsin K under acidic conditions. Similar results were found with 3-bromo-3'-hydroxybenzophenone thiosemicarbazone (8). Compounds 8 and 1 inhibited the in vitro type I collagenase activity of cathepsin L in a time-dependent manner and type IV collagenase activity of cathepsin K. Analogs, 1, 8, 156, 157, and 168 were also used in cell studies. These compounds were able to delay cell migration and cell invasion in MDA-MB-231 cells, a type of carcinoma breast cancer cell line. It was determined that cell invasion and cell migration were inhibited in a concentration dependent manner. Lastly, analog 17 was also found to be a slow reversible, slow binding, competitive inhibitor of cruzain. This compound was also able to inhibit the collagenase activity of recombinant cruzain when human type I collagen was used as a substrate. Kinetic studies and molecular modeling indicate the best thiosemicarbazone inhibitors form a reversible covalent bond with each enzyme. 2013-05-15T00:00:00Z http://hdl.handle.net/2104/8518 Design and synthesis of non-peptidic thiosemicarbazone derivatives as inhibitors of cathepsins L and K. Song, Jiangli. Cathepsin L, a member of the lysosome cysteine proteases, is ubiquitously expressed in tissue and is responsible for protein turnover. An overexpression of cathepsin L by certain tumor types is associated with enhanced tumor expansion through degradation of the extracellular matrix and the invasion and migration of cancer cells. Therefore, the inhibition of cathepsin L has emerged as a therapeutic strategy against metastatic cancer. Cathepsin K is involved in osteoclastic bone resorption because it is selectively expressed in osteoclasts and is capable of degrading bone matrix. In this project, a small library of thiosemicarbazone derivatives containing thiochromanone, 2,3-dihydroquinoline-4-one, and dibenzoylbenzene scaffolds has been successfully designed and synthesized as potential inhibitors of cathepsin L and other homologous cathepsins (B and K). Through a collaborative study, ten compounds from this library were found to be potent inhibitors (IC50 < 300 nM) of cathepsin L, and nine compounds were potent inhibitors of cathepsin K. None of these inhibitors showed activity against cathepsin B. For example, 6,7-difluorothiochromanone thiosemicarbazone 4 (IC50 = 46 nM) was the most potent inhibitor against cathepsin L from this group, while the 6-trifluoromethyl derivative 28 (IC50 = 21 nM) was the most potent inhibitor against cathepsin K. Structure activity relationship (SAR) studies centered on the thiochromanone thiosemicarbazone scaffold demonstrated that electron-withdrawing functionalities, incorporated primarily at the 6-position of the thiochromanone scaffold, showed good inhibition against cathepsin L in comparison to related analogues bearing electron-donating groups. Collectively, these results expand the known SAR regarding molecular structures and their inhibitory activity against cathepsins L and K. 2012-11-29T00:00:00Z http://hdl.handle.net/2104/8482 Targeting the tumor microenvironment through the design and synthesis of potent, small-molecule, anticancer agents. George, Clinton S. Targeting the vascular network that supplies a tumor with oxygen and nutrients is a viable methodology for the treatment of cancer. With the discovery of the combretastatin extended family of compounds in the late 1970's and early 80's, there has been an accelerated interest in vascular targeting as a possible cancer treatment methodology. Two natural products, combretastatin A-1 (CA1) and combretastatin A-4 (CA4) interfere with the tubulin-microtubule protein system in the endothelial cells lining tumor vasculature, ultimately resulting in vessel damage. A new class of compounds shares similar functional group motifs and structural orientations to CA4, but is based on a benzosuberene core structure. These benzosuberene analogues incorporate a [6,7] fused ring system with an aryl ring attached at the 5-position. The initial discovery in the Pinney Research Group of the benzosuberene lead compound 1-hydroxy-2-methoxy-5-(3',4',5'-trimethoxyphenyl)benzocyclohept-5-ene 1, has led to research focused on the development of an efficient strategy for synthesizing [6,7] fused ring systems and the preparation of new, diversely functionalized benzosuberene analogues. New derivatives include regioisomeric incorporations of methoxy and hydroxy groups, along with the inclusion of nitro, amine, and halogen substituents. Bioreductively activated prodrug triggers, as well as [6,8] and [5,7] fused ring systems are also included in the library of compounds developed. Initial biological evaluation of these benzosuberene compounds provide interesting results. These compounds were evaluated for cytotoxicity against three human cancer cell lines, and for their ability to inhibit the assembly of tubulin into microtubules. Among the library of compounds screened, several analogues were discovered that are comparable in activity to CA4 and CA1. For example, 1,2-dihydroxy-5-(3',4',5'-trimethoxyphenyl)-benzosuber-5-ene 20 and 1,2-dimethoxy-5-(3',4',5'-trimethoxyphenyl)-benzosuber-5-ene 22 are strongly cytotoxic against the NCI-H460 human cell line (GI50 = 0.410 and 0.479 μM respectively). Collectively, the structure-activity relationship (SAR) knowledge gained from these compounds will guide the design of new benzosuberene-based anticancer agents. 2012-08-08T00:00:00Z