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Browsing by Author "Khanna, May"
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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 Discovery and characterization of small molecules that target the Ral GTPase(Nature Publishing Group, 2014-11-20) Yan, Chao; Liu, Degang; Li, Liwei; Wempe, Michael F.; Guin, Sunny; Khanna, May; Meier, Jeremy; Hoffman, Brenton; Owens, Charles; Wysoczynski, Christina L.; Nitz, Matthew D.; Knabe, Eric W.; Brautigan, David L.; Paschal, Bryce M.; Schwartz, Martin A.; Jones, David; Ross, David; Meroueh, Samy O.; Theodorescu, Dan; Department of Biochemistry & Molecular Biology, IU School of MedicineThe Ras-like GTPases RalA and B are important drivers of tumor growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here, we used protein structure analysis and virtual screening to identify drug-like molecules that bind a site on the GDP-form of Ral. Compounds RBC6, RBC8 and RBC10 inhibited Ral binding to its effector RalBP1, Ral-mediated cell spreading in murine fibroblasts and anchorage-independent growth of human cancer cell lines. Binding of RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasma resonance and 15N-HSQC NMR. RBC8 and BQU57 show selectivity for Ral relative to Ras or Rho and inhibit xenograft tumor growth similar to depletion of Ral by siRNA. Our results show the utility of structure-based discovery for development of therapeutics for Ral-dependent cancers.Item Discovery of Novel Regulators of Aldehyde Dehydrogenase Isoenzymes(2011-05) Parajuli, Bibek; Kimble-Hill, Ann C.; Khanna, May; Ivanova, Yvelina; Meroueh, Samy; Hurley, Thomas DOver the past three years we have been involved in high-throughput screening in an effort to discover novel small molecular modulators of aldehyde dehydrogenase (ALDH) activity. In particular, we have been interested in both the activation and inhibition of the three commonly studied isoenzymes, ALDH1A1, ALDH2 and ALDH3A1, as their distinct, yet overlapping substrate specificities, present a particularly difficult challenge for inhibitor discovery and design. Activation of ALDH2 has been shown to benefit cardiovascular outcome following periods of ischemia and renewed interest in specific inhibition of ALDH2 has application for alcohol aversion therapy, and more recently, in cocaine addiction. In contrast, inhibition of either ALDH1A1 or ALDH3A1 has application in cancer treatments where the isoenzymes are commonly over-expressed and serve as markers for cancer stem cells. We are taking two distinct approaches for these screens: in vitro enzyme activity screens using chemical libraries and virtual computational screens using the structures of the target enzymes as filters for identifying potential inhibitors, followed by in vitro testing of their ability to inhibit their intended targets. We have identified selective inhibitors of each of these three isoenzymes with inhibition constants in the high nanomolar to low micromolar range from these screening procedures. Together, these inhibitors provide proof for concept that selective inhibition of these broad specificity general detoxication enzymes through small molecule discovery and design is possible.Item Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain(Lippincott, Williams & Wilkins, 2018-10) François-Moutal, Liberty; Dustrude, Erik T.; Wang, Yue; Brustovetsky, Tatiana; Dorame, Angie; Ju, Weina; Moutal, Aubin; Perez-Miller, Samantha; Brustovetsky, Nickolay; Gokhale, Vijay; Khanna, May; Khanna, Rajesh; Pharmacology and Toxicology, School of MedicineWe previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.Item Sustained relief of ongoing experimental neuropathic pain by a CRMP2 peptide aptamer with low abuse potential(Wolters Kluwer, 2016-09) Xie, Jennifer Y.; Chew, Lindsey A.; Yang, Xiaofang; Wang, Yuying; Qu, Chaoling; Wang, Yue; Federici, Lauren M.; Fitz, Stephanie D.; Ripsch, Matthew S.; Due, Michael R.; Moutal, Aubin; Khanna, May; White, Fletcher A.; Vanderah, Todd W.; Johnson, Philip L.; Porreca, Frank; Khanna, Rajesh; Anesthesia, School of MedicineItem 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.