Biomedical Sciences ETDs

Jacob Agola

Publication Date

12-1-2011

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.

Keywords

Rab, Rho, Rac, Cdc42 and Ras GTPases; drug discovery; fluorescent GTP and GDP; enzyme kinetics; GEF; flow cytometry, ovarian cancer, competitive and allosteric inhibitors

Sponsors

University of New Mexico (UNM), National Science Foundation (NSF) and National Institutes of Health (NIH)

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Hudson, Laurie

Second Committee Member

Prossnitz, Eric

Third Committee Member

Buranda, Tione

Fourth Committee Member

Sklar, Larry

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