Date of Award
Ph.D. in Pharmaceutical Sciences
Tracy A. Brooks
Christopher R. McCurdy
John M. Rimoldi
DNA G-quadruplexes (G4s) have gained interest in modern drug discovery efforts. The majority of G4 research, including the mass of this research, heavily focuses on cancer therapeutic development, as G4s are more pronounced in oncogenic promoters compared to the rest of the genome, our efforts extend towards neuroprotection as well. In particular, this work covers G4s in the promoters of MYC, VEGF, kRAS, and Bcl-2. MYC and VEGF harbor the two most well described promoter G4s to date, and their stabilization cultivates transcriptional silencing. Downregulation of MYC reduces cell growth and alters cell energetics; it is promising for a variety of cancer types, including lymphomas and leukemias. VEGF downregulation modulates angiogenesis, therapeutically benefiting most solid tumors. Lesser described G4s, kRAS and Bcl-2, act as transcriptional silencers and activators, respectively. kRAS is highly deregulated in 30% of cancers, and its downregulation can decrease tumor cell proliferation and survival. Decreases in Bcl-2 expression result in pro-apoptotic signaling. However, Bcl-2 G41 stabilization can cause transcriptional activation, and its upregulation reduces apoptosis, which is of therapeutic benefit for neurodegenerative diseases. The current study examines (a) the effect of epigenetic modification on VEGF G4 stability, (b) a variety of osmolytes and crowding co-solvents for their ability to recapitulate the physiological structure in single-stranded ex vivo conditions, (c) characterizing and targeting the biologically relevant G4 in the kRAS core promoter for a novel approach to targeting kRAS driven cancers, specifically pancreatic cancer, and (d) targeting one of four G4s (G41) within the Bcl-2 promoter for neuroprotection. For all of the studies, significant changes in G4 structures, including loop directionality and number of competing isoforms, were examined by electronic circular dichroism. Electromobility shift assays differentiated inter- and intra-molecular structures, further distinguishing the distribution of isoforms. Other studies included chemical footprinting, FRET compound screening, luciferase, and in vitro cytotoxicity. This comprehensive understanding of the physiological conditions regulating G4 stability and function using co-solvents to examine the effects of small molecules ex vivo will best inform future drug discovery efforts on how to better predict active hits for in vitro evaluation for these and other promoters.
Morgan, Rhianna Kyelle, "G-Quadruplexes As Molecular Targets For Transcriptional Control" (2017). Electronic Theses and Dissertations. 722.