Human Telomerase Holoenzyme Assemblage as an Anticancer Drug Target Public Deposited

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  • March 20, 2019
Creator
  • Keppler, Brian
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
Abstract
  • Telomerase is an RNA-dependent DNA polymerase that extends the 3' ends of linear chromosomes. Almost 90% of all cancers require telomerase activity in order to maintain their immortal phenotype. This thesis describes a novel platform for human telomerase inhibition as an anticancer approach. Preliminary studies included testing the feasibility of perturbing proper human telomerase assemblage as a means of inhibiting enzymatic activity. This methodology was validated using oligonucleotides targeted at specific domains of the telomerase RNA subunit (hTR), which were found to inhibit telomerase activity in a direct assay by preventing the association of hTR with the telomerase protein subunit (hTERT) when added prior to assembly. Following these proof-of-principle studies, this approach was further authenticated with the use of known nucleic acid-binding ligands. Various compounds, including DNA minor groove binders and aminoglycoside antibiotics, were found to decrease telomerase activity to a greater extent when added prior to hTR/hTERT assembly as compared to their addition after hTR an hTERT were allowed to associate. A small library of compounds including various tanshinone natural products and some synthetic derivatives was tested for telomerase inhibition using the pre- and post-assembly parallel screen. Results indicate that the most potent inhibitor was tanshinone II-A. This compound completely inhibited activity prior to assembly, however, could only partially inhibit activity after assembly indicating that its mode of action could be preventing telomerase assemblage. Studies here also further characterize and elucidate the roles of the telomerase-associated protein Hsp90 and reveal that the presence of Hsp90 is unremittingly required in order to maintain telomerase in an active conformation. Results show that the N-terminus of Hsp90 may be involved in preparing telomerase for telomeric primer loading while the role of the C-terminus may be to stabilize the telomerase holoenzyme complex. This new technique of identifying novel telomerase inhibitors was converted into a high-throughput format. Scintillation proximity assay technology was utilized in order to design and develop a screen capable of identifying compounds that perturb a specific interaction between hTR and hTERT. The assay was optimized and validated using an oligonucleotide previously shown to preclude this interaction. These studies indicate that human telomerase assemblage is a viable anticancer drug target. The further development and optimization of telomerase assemblage exploitation methods could lead to a new class of clinically relevant telomerase inhibitors.
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  • In Copyright
Advisor
  • Jarstfer, Michael
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2006
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