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Assessment of functional characteristics of small GTPases using small molecules


Please use this identifier to cite or link to this item: http://hdl.handle.net/1928/17461

Assessment of functional characteristics of small GTPases using small molecules

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Title: Assessment of functional characteristics of small GTPases using small molecules
Author: Agola, Jacob
Advisor(s): Wandinger-Ness, Angela
Committee Member(s): Hudson, Laurie
Prossnitz, Eric
Buranda, Tione
Sklar, Larry
Department: University of New Mexico. Biomedical Sciences Graduate Program
Subject: Rab, Rho, Rac, Cdc42 and Ras GTPases; drug discovery; fluorescent GTP and GDP; enzyme kinetics; GEF; flow cytometry, ovarian cancer, competitive and allosteric inhibitors
LC Subject(s): Guanosine triphosphatase.
Nonsteroidal anti-inflammatory agents.
Enzyme kinetics.
Degree Level: Doctoral
Abstract: Rab and Rho subfamilies of GTPases are functionally linked to intracellular trafficking and organization of the cytoskeleton respectively. Despite their roles, use of small molecule inhibitors or activators to map the functionality of these GTPases remains largely underexplored due to lack of suitable compounds. In this dissertation, we report on the functional characterization of Rab7 and Rac1 GTPases using small molecules. 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7- dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem #: CID1067700) has been used to characterize Rab7. Using bead based flow cytometry, CID1067700 was found to have significant inhibitory potency on Rab7 nucleotide binding with a respective inhibitory efficacy of 80% for BODIPY-GTP and 60% for BODIPY-GDP binding. Rac1 has been functionally characterized by non-steroidal anti-inflammatory drug (NSAID), R-Naproxen in the context of ovarian cancer. R-Naproxen isoform functionally inhibited Rac1 in the cell lines assayed relative to S-Naproxen and structurally similar 6-methoxy-2-naphthylacetic Acid (6-MNA). Inhibition is based on interference with membrane distribution of Rac1 rather than overall protein levels. Taken together, this study has identified the first competitive GTPase inhibitor (CID1067700) and also demonstrated the potential utility of the compound for dissecting GTPase enzymology. The study has also shown that R-Naproxen blocks activation of Rac1 small GTPase in ovarian cancer cells with implications for the inhibition of ovarian cancer cell proliferation, migration, and invasion.
Graduation Date: December 2011
URI: http://hdl.handle.net/1928/17461

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