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Browsing by Author "Jo, Inha"
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Item BIOCHEMICAL CHARACTERIZATION OF SMALL MOLECULES TARGETING RAL GTPASE(Office of the Vice Chancellor for Research, 2012-04-13) Ishikawa, Megan; Khanna, May; Jo, Inha; Meroueh, SamyThe Ral subfamily of GTPases consists of highly similar RalA and RalB isoforms that participate in diverse cellular functions including endocytosis, exocytosis, actin cytoskeletal dynamics, and transcription. A large body of evidence has implicated Ral GTPases with tumor cell growth, migration, and angiogenesis in bladder, prostate, lung, and pancreatic cancer. The purpose of this project was to target the activity of Ral GTPases and their association with effector proteins through the identification of small molecule inhibitors that block this interaction. In order to accomplish this, both direct binding to RalB as well as disruption of protein-protein interaction were investigated. The top 200 compounds from a larger computational library of 500,000 compounds targeting the RalBP1 binding site on RalB were tested. Differen-tial scanning fluorimetry (DSF) was used to measure the degree of direct binding between compound and protein through thermal melting shift. To measure disruption between RalB and RalBP1 by small molecules, a novel enzyme-linked immunosorbent assay (ELISA) was developed. Identification of a few key compounds binding to RalB as well as optimization of an ELISA assay for RalBP1 was accomplished. Further direction of this project would be to utilize the ELISA assay to test inhibition of the protein-protein interac-tion between RalB and RalBP1 using the top compounds from the DSF trials.Item Virtual Screening Targeting the Urokinase Receptor, Biochemical and Cell-Based Studies, Synthesis, Pharmacokinetic Characterization, and Effect on Breast Tumor Metastasis(American Chemical Society, 2011-10-27) Wang, Fang; Li, Jing; Sinn, Anthony L.; Knabe, William Eric; Khanna, May; Jo, Inha; Silver, Jayne M.; Oh, Kyungsoo; Li, Liwei; Sandusky, George E.; Sledge, George W.; Nakshatri, Harikrishna; Jones, David R.; Pollok, Karen E.; Meroueh, Samy O.Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.