Date of Degree
PhD (Doctor of Philosophy)
Medicinal and Natural Products Chemistry
David L. Roman
First Committee Member
Jonathan A Doorn
Second Committee Member
Michael W Duffel
Third Committee Member
Michael A Spies
Fourth Committee Member
Rory A Fisher
Regulator of G-protein Signaling (RGS) proteins temporally regulate the G protein signaling cascades initiated by GPCR activation. Reports have established dysregulation of RGS expression in a variety of disease states including several cancers. Additionally, use of genetic ablation techniques has implicated RGS proteins in a variety of other disease states through the native action of the RGS i.e. not a consequence of dysregulation of RGS expression. Therefore identification and optimization of small molecule lead compounds that alter RGS protein function has emerged as a promising therapeutic strategy.
In this thesis, we use high throughput screening to interrogate small molecule libraries targeting two RGS proteins, RGS6 and RGS17. RGS6 has been reported as an essential mediator of doxorubicin induced cardiotoxicity, alcohol induced cardio and hepatotoxicity, anxiety, depression, and alcohol dependence. RGS17 has largely been implicated in a variety of cancer pathogenesis, with reported over expression in prostate, lung, breast, and hepatocellular carcinomas.
Chapter 2 of this work focuses on the screening efforts targeting RGS6. Three separate screening campaigns interrogating over 20K compounds led to the identification of 3 small molecules that inhibit the RGS6: Gαo protein protein interaction with appreciable selectivity over control assays. The development of a cell based protein interaction assay is discussed, and the compounds were investigated using this system. All compounds tested did not appreciably alter signal over control, meaning that the cellular activity of these compounds remains ambiguous.
Chapter 3 details the screening and follow up efforts targeting RGS17. The primary screening and/or follow up of four separate screening campaigns interrogating over 110K compounds is discussed. In total, 10 identified leads and a panel of analogs were subjected to significant follow up evaluation. All compounds were found to be cysteine dependent. The second generation RGS17 inhibitors (UI series) were determined to be both cytostatic and cytotoxic against lung and prostate cancer cell lines in culture, although whether this is due to RGS17 dependent mechanisms or due to general promiscuity of the compounds remains to be determined. Lead compounds from a library provided by the NCI were found to have cellular activity and were subjected to an investigation of structure activity relationships via commercially available compounds. The active form of three of these compounds was found to be a degradation product, which is likely due to decomposition of furan or methyl furan moieties that these compounds shared. One compound demonstrated robust SAR which allowed for the generation of schemes detailing putative inhibitory mechanisms. Finally, the role of RGS17 in the transition from epithelial to mesenchymal phenotypes is investigated. RGS17 was found to cause a sub population of PC3 cells to shift to mesenchymal phenotype, indicating that RGS17 may indeed play a role in this transition.
Chapter 4 focuses on efforts to investigate variable potencies of published RGS4 inhibitors against a panel of RGS proteins, with the goal of gleaning insight in to structural characteristics that influence the inhibitability of RGS proteins. Most compounds tested were found to be more potent inhibitors of RGS14 rather than RGS4 in biochemical assays. We developed the NanoBit protein complementation assay to assess the interaction of RGS proteins with either Gαi1 or Gαq in a cellular context, and used this system to investigate compound selectivity in a cellular context. The compounds tested showed selectivity for RGS2, RGS4, and RGS14 over the other RGS proteins tested. The structural differences between the RGS proteins is discussed.
Chapter 5 focuses on the future directions the lab may take with respect to the projects outlined in the previous chapters. This includes the screening of more targeted libraries or even virtual screening for RGS6, the development of in vivo assessment tools for RGS17, and an expanded structural examination of RGS proteins including NMR and crystal structure analysis. Additionally, the development of the NanoBit system to interrogate RGS protein interactions that are not RGS: Gα interactions is discussed.
Cancer remains the second leading cause of death in the United States. The complexity of cancer necessitates a constant investigation of cell signaling pathways essential to cancer survival, and the identification of new protein targets for therapeutic development. One family of proteins, Regulator of G Protein Signaling or RGS proteins, has been identified as playing a role in multiple cancers as well as other disease states. The focus of this work is on the identification of chemical compounds that can inhibit the function of two RGS proteins, RGS6 and RGS17.
RGS6 has been reported as essential in the development of cardiotoxicity in cancer patients who are treated with the chemotherapeutic doxorubicin. This cardiotoxicity imposes a lifetime dose limit for this drug, one of the most successful chemotherapeutics available. Additionally, RGS6 has been reported as target for the treatment of anxiety, depression, and alcohol dependence. For RGS17, this protein is reported as overexpressed in prostate, lung, breast, and hepatocellular carcinomas, which leads to increased tumor growth and metastatic potential.
We use robotic systems to investigate tens of thousands of compounds to identify compounds that inhibit the function of these two proteins. This work details the screening effort, and the validation of compounds discovered including assessing activity in cell culture. We found 2-3 compounds that inhibit RGS6 and 10-20 compounds that inhibit RGS17. We also investigated the selectivity of these compounds for the intended target and investigated how the compounds are doing what they do.
Alcoholism, Cancer, Depression, G protein, High Throughput Screening, RGS
xviii, 203 pages
Includes bibliographical references (pages 194-203).
Copyright © 2017 Christopher Ralph Bodle
Bodle, Christopher Ralph. "Identification of small molecule inhibitors of regulator of G protein signaling proteins for pretherapeutic development for treatment of multiple pathologies." PhD (Doctor of Philosophy) thesis, University of Iowa, 2017.