Date on Master's Thesis/Doctoral Dissertation

12-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Trent, John O.

Committee Co-Chair (if applicable)

Chaires, Jonathan B.

Committee Member

Chaires, Jonathan B.

Committee Member

Clark, Barbara J.

Committee Member

Ellis, Steven R.

Committee Member

Sabo, Thomas M.

Author's Keywords

G-quadruplex; DNA; small-angle X-ray scattering; drug discovery; molecular dynamics; hydrodynamics

Abstract

There is now substantial evidence that guanine-rich regions of DNA form non-B DNA structures known as G-quadruplexes in cells. G-quadruplexes (G4s) are tetraplex DNA structures that form amid four runs of guanines which are stabilized via Hoogsteen hydrogen bonding to form stacked tetrads. DNA G4s have roles in key genomic functions such as regulating gene expression, replication, and telomere homeostasis. Because of their apparent role in disease, G4s are now viewed as important molecular targets for anticancer therapeutics. To date, the structures of many important G4 systems have been solved by NMR or X-ray crystallographic techniques. Small molecules developed to target these structures have shown promising results in treating cancer in vitro and in vivo, however, these compounds commonly lack the selectivity required for clinical success. There is now evidence that long single-stranded G-rich regions can stack or otherwise interact intramolecularly to form G4-multimers, opening a new avenue for rational drug design. For a variety of reasons, G4 multimers are not amenable to NMR or X-ray crystallography. In the current dissertation, I apply a variety of biophysical techniques in an integrative structural biology (ISB) approach to determine the primary conformation of two disputed higher-order G4 systems: (1) the extended human telomere G-quadruplex and (2) the G4-multimer formed within the human telomerase reverse transcriptase (hTERT) gene core promoter. Using the higher-order human telomere structure in virtual drug discovery approaches I demonstrate that novel small molecule scaffolds can be identified which bind to this sequence in vitro. I subsequently summarize the current state of G-quadruplex focused virtual drug discovery in a review that highlights successes and pitfalls of in silico drug screens. I then present the results of a massive virtual drug discovery campaign targeting the hTERT core promoter G4 multimer and show that discovering selective small molecules that target its loops and grooves is feasible. Lastly, I demonstrate that one of these small molecules is effective in down-regulating hTERT transcription in breast cancer cells. Taken together, I present here a rigorous ISB platform that allows for the characterization of higher-order DNA G-quadruplex structures as unique targets for anticancer therapeutic discovery.

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