Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry, Biochemistry

Date of Defense

4-19-2018

Graduate Advisor

Dr. Cynthia Dupureur

Committee

Dr. James Bashkin

Dr. Wesley Harris

Dr. Michael Nichols

Abstract

While vaccines exist for the some of the most problematic strains of human papillomavirus (HPV), a double stranded DNA virus, there is currently no cure. HPV remains one of the most commonly sexually transmitted infections and is responsible for virtually all cervical cancers and genital warts. Natural products Distamycin A and netropsin have inspired the hairpin Nmethylpyrrole (Py)/N-methylimidazole (Im) polyamides (PAs) studied here. The larger hairpin PAs, designed to bind to sites of 10 or more DNA bp, have been shown to be effective antivirals against oncogenic HPV strains 16, 18, and 31, while smaller hairpin PAs are not. Despite significant differences in potencies among the antiviral PAs tested, the PAs bind to DNA with similar binding affinity (Kd). Ample evidence has shown dissociation rate constants (koff) may be a better indicator of drug efficacy than Kd. While Kd is a function of association rate constant (kon) and koff, respectively (Kd = koff/kon), few studies have focused on the DNA binding kinetics of large hairpin PAs.

We are using fluorescence and CD spectroscopy to characterize Kds, obtain DNA binding kinetic rate constants, and determine binding stoichiometries as a function of PA size. K= remains tight (low nM) for all PAs tested (6-20-rings) with our fluorescence assay, which is consistent with what is seen with other methods. The large PAs are characterized by slow DNA dissociation rates with half-lives ranging from 20-30 min, and dissociation slows as the size of PAs increases. Slow dissociation rates are likely the source of the difference in antiviral behavior. Association time courses for a 14-ring PA indicate that >1 equivalent of PA is binding to the DNA. Further supporting this claim, fluorescence and CD spectroscopic experiments indicate that saturation of the DNA does not occur until 2 or more equivalents of PA are added. The slow dissociation of multiple equivalents of PA bound to DNA may cause a large and prolonged disruption in DNA conformation, which in turn elicits or alters the DNA damage response, which is an integral part in the antiviral mechanism of large antiviral hairpin PAs and of the lifecycle of HPV.

Available for download on Thursday, April 23, 2020

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