The sequencing of the human genome suggests that transcription factors (TFs) make up one of the largest classes of human proteins, revealing that there are over 2000 genes that code for transcription factors. The pivotal roles of TFs in cell biology become quite apparent when one or more of these regulatory mechanisms becomes mutated or altered. For example, the androgen receptor (AR) transcription factor plays a pivotal role in prostate carcinogenesis and progression. Additionally, the inflammatory response of the NF-κB transcription factor proteins results in the transcription of many genes, which play pivotal roles in carcinogenesis. There are several approaches to modulate and study transcription factor activity and biochemistry. Utilizing cis element DNA decoys to sequester TFs is one approach to directly modulate transcription factors. Introducing these synthetic double-stranded DNA decoys containing TF binding sites into cells effectively sequesters TFs and inhibits their target gene expression. Over the past couple of decades, numerous reports have validated utilizing this approach. For example, a phosphorthioate STAT3 DNA decoy has entered the “first-in-human” Phase 0 clinical trials for the treatment of head and neck squamous cancer. STAT3 expression and cell viability was reduced in the head and neck cancers injected with the decoy compared to the saline control. Combining the spatial and temporal resolution of caging technology with the DNA decoy strategy for the inhibition of transcription factor activity can yield an approach for the very precise ability to photochemically regulate gene expression, which has potential as a therapeutic agent and tool for probing biological pathways. This thesis will focus on efforts to develop several novel DNA-based and small molecule-based probes to investigate the biochemistry of TFs and their signaling pathways. Chapter 2 discusses the synthesis and characterization of caged DNA decoys that target the Androgen Receptor (AR). Caged DNA decoys successfully captured AR in LNCaP lysate when irradiated with light. Chapter 3 introduces the complement to caging technology, which is catch and release DNA decoys (CRDDs). CRDDs capture transcription factors, by binding and sequestering them, and then a pulse of light photochemically destroys the CRDD, permitting release of the TF. Several 7-nitroindole (7-NI, 1.47) nucleobase mimics were incorporated into NF-κB-directed DNA decoys, which still allowed the capture of the p50-p65 NF-κB proteins. Irradiation with 350 nm light drives the release of the p50-p65 NF-κB. The capture and photochemical release of an endogenous transcription factor is demonstrated for the first time. Chapter 4 continues the work of Chapter 3 by developing second-generation nucleobase mimics for use in CRDDs. Addition of molecular recognition properties on a photo-responsive monomer is hypothesized to increase binding affinity to capture endogenous TFs. 8-Nitroguanosine contains this added molecular recognition, is more stable within duplex DNA, and also displayed similar photochemical depurination properties. Chapter 5 outlines work developing photoswitchable nucleobases that transpose their hybridization properties upon photolysis. Chapter 6 highlights work to determine the mechanistic NF-κB inhibitory properties of several Cryptocaryone analogues, which were found to inhibit the NF-κB translocation to the nucleus. Appendix A focuses on the characterization of the enantioselectivity of guanosine monophosphate synthetase (GMPS), a crucial enzyme in nucleotide biosynthesis.
University of Minnesota Ph.D. dissertation. February 2016. Major: Medicinal Chemistry. Advisor: Daniel Harki. 1 computer file (PDF); xiii, 260 pages.
Synthesis of Photoresponsive Nucleosides and Their Incorporation into Oligonucleotides: Targeting Androgen Receptor and NF-κB Transcription Factors.
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