Brandi M. Baughman Abstract - ETD Index Page

Brandi M. Baughman
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Doctoral Dissertation
Brandi M. Baughman
Brandi.M.Baughman@gmail.com
Structural Identification and Applications of
Novel Fluorescent Probes in Chemical Biology
Doctor of Philosophy
Integrated Program in Biomedical Sciences
Molecular Therapeutics and Cell Signaling
Thomas R. Webb, Ph.D.
twebb13@uthsc.edu
Donald Bashford, Ph.D.
Richard Lee, Ph.D.
Stephen White, D.Phil.
Jie Zheng, Ph.D.
Endonuclease, Fluorescence, Influenza
World-Wide Web Access
Graduation Date: December 2012
Abstract
Fluorescence spectroscopy is one of the most widely used tools in drug discovery; however,
specific criteria for compound fluorescence are not well defined. While a highly sensitive
method of detection, fluorescence-based sensing is susceptible to assay interference due to
intrinsically fluorescent compounds. Current methods of fluorescence prediction do not consider
high-throughput screening (HTS) requirements and therefore are not applicable for filtering large
chemical databases. The studies described herein aim to establish structural and electronic
criteria for fluorescent emission in HTS, identify structure-fluorescence relationships (SFR) for
novel sets of fluorophores, and develop fluorescence-based assays using an explicit empirical
definition of fluorescence. Our working definition of fluorescence caters to the unique
environment of HTS and is described as BGA: bright (≥5.0% fluorescence intensity of

fluorescein at the same concentration), green (emission at 535 nm ± 25 nm upon excitation at
485 nm ± 20 nm), and aqueous (fluorescence observed in a buffered solution at a specific
concentration). Implementing a decision tree learning-based algorithm based on calculable
molecular properties, we were able to predict BGA fluorescence with 93% accuracy for a sample
library of compounds (N = 2,231). From this sample library, two novel fluorophores were
identified. Spectral evaluation of these fluorophores resulted in the validation of a dual
fluorophore with red and green emissions and a bright green fluorophore with chemical and
fluorescent stability in varying solvents and bioavailability in zebrafish. Finally, we have
developed a high-throughput fluorescence polarization (FP) assay, utilizing a novel fluoresceinlabeled
compound (K d = 0.378 µM) and a PA N construct, to identify small molecules that bind
to the PA N endonuclease active site of influenza virus. This FP assay provides direct target
validation information for active compounds, is suitable for HTS and fragment-based screening
studies, and has resulted in the identification of novel endonuclease inhibitors. Our work
suggests that with a basic understanding of the fundamental requirements for fluorescence in an
HTS environment, SFR can be predicted, and interference due to intrinsically fluorescent
compounds can be controlled within assay settings.
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Revised 21 March 2013