Targeting Mutant TERT Promoter in Glioblastoma to inhibit Replicative Immortality
Metell, Amanda, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Bushweller, John, MD-MPHY Mole Phys & Biophysics, University of Virginia
Glioblastoma (GBM) is the most common and malignant brain tumor, with a life expectancy after prognosis of only 15 months. Current therapies for this malignant disease are largely invasive and ineffective. Telomerase is an enzyme that is responsible for maintaining the length of telomeres on DNA, which allows for the ongoing replication of cells. While there are a number of potential targets for therapeutics for GBM, targeting TERT (catalytic subunit of telomerase) seems very promising since mutations in the promoter of TERT increase its expression and are observed in 83% of primary GBMs. Targeting TERT in GBM cells would alter telomerase activity, denying the cancer cells replicative immortality. The promoter mutation in TERT in GBMs results in the loss of binding to the ETS (E26 transformation-specific) family transcription factor, ELF1/2, with replacement by the ETS family transcription factor, GA-binding protein (GABP) which results in increased TERT expression. GABP is a heterodimeric transcription factor consisting of a GABPα DNA binding subunit and a GABPβ transactivation subunit. Since GABPα and GABPβ must bind to each other to increase TERT level, this project aimed to develop a protein-protein interaction inhibitor of GABPα and GABPβ as a therapy for GBM. In this study, the behavior of GABPα and GABPβ was explored with and without the addition of small molecules. First, a FRET assay was developed to monitor the binding by fusing the GFP derivatives, Cerulean and Venus, to the relevant portions of GABPα and GABPβ. With this assay a binding constant as well as IC50 values were generated for the proteins and compounds. To confirm which proteins the compounds were binding, a mutated form of GABPβ (absence of cysteines) was expressed and purified. Lastly, GABPβ, upon the addition of several compounds, was analyzed through 15N-1H HSQC NMR experiments.
The work in this study has identified several compounds with IC50 values of less than 1 mM. These compounds represent the building blocks of a potent inhibitor against GABPα and GABPβ. In this study, it was also shown that the cysteine reactive compounds are not binding to the cysteine located on GABPβ. Lastly, this study shows that based on the 15N-1H HSQCs of compounds when bound to GABPβ, the spectrum becomes more resolved, insinuating that these compounds stabilize GABPβ upon binding, or that GABP undergoes a conformational change upon binding to compounds.
MS (Master of Science)
TERT, Glioblastoma, GABP
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