Woo, Christina MCheng, Steven Shiang2026-01-1320252025-06-052025Cheng, Steven Shiang. 2025. Investigation of small molecule modulators of O-GlcNAc. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32041479https://dash.harvard.edu/handle/1/42725186In the central dogma of molecular biology, DNA is transcribed to RNA, which is spliced and then translated into proteins, which execute various biological functions and themselves are regulated by post-translational modifications. O-GlcNAc is an essential and ubiquitous monosaccharide post-translational modification involved in many major cellular processes. O-GlcNAc is installed and removed by a pair of enzymes, the writer O-GlcNAc transferase (OGT) and the eraser O-GlcNAcase (OGA). The cell tightly regulates these enzymes and thus O-GlcNAc homeostasis through a highly conserved post-transcriptional mechanism. Through this detained intron feedback mechanism, OGT and OGA levels are inversely adjusted to maintain O-GlcNAc levels. For example, if O-GlcNAc is elevated, the cell responds by downregulating OGT and upregulating OGA to diminish O-GlcNAc, and vice versa in the case that O-GlcNAc is lowered. Here, I present work detailing the development of assays to study O-GlcNAc and its cycling enzymes in vitro and in cells and the application of these assays to high-throughput screening, leading to the discovery of small molecule splicing modulators that can disrupt O-GlcNAc homeostasis. In Chapter 1, I review the O-GlcNAc field, covering the multitude of protein biochemistry, medicinal chemistry, and chemical biology studies performed since the initial discovery of O-GlcNAc over four decades ago. This chapter also provides a timely update to clinical efforts to therapeutically modulate O-GlcNAc, primarily focused on OGA inhibition for the treatment of neurodegenerative diseases such as Alzheimer’s disease. In addition to highlighting the major fundamental discoveries within the O-GlcNAc field, I also emphasize opportunities for further research into the basic biology of O-GlcNAc and its cycling enzymes and the potential therapeutic application of these findings. In Chapter 2, I discuss our assay development efforts towards discovery of OGT ligands. We adapted and developed protocols to purify OGT constructs and subsequent assays to characterize protein—ligand binding interactions including fluorescence polarization (FP), time-resolved Förster resonance energy transfer (TR-FRET), and differential scanning fluorimetry (DSF). We additionally discuss efforts to use DSF as a high-throughput screening assay to discover OGT binders from a bioactive compound library and a screening informer set and subsequent validation of these hits. While ultimately no hits were discovered to be selective OGT binders from these screens, the protocols developed here will be valuable for future efforts to discover OGT ligands through similar assays. In Chapter 3, I describe three parallel screening campaigns employing the Broad Institute Drug Repurposing Hub. Specifically, we screened this library using in vitro enzymatic assays against OGT and OGA and using a cellular reporter of OGT splicing in a high-content microscopy format to understand how clinically relevant compounds and pathways may intersect with O-GlcNAc homeostasis. We describe the optimization and miniaturization of these assays and the subsequent validation of hits from these screening efforts. I report that the dopamine receptor agonist piribedil and the AKT inhibitor GSK690693 have previously unannotated OGA inhibitor activity, and that GSK690693 and the ROCK inhibitor Y-33075 are able to disrupt O-GlcNAc homeostasis in cells, downregulating both OGT and OGA. In Chapter 4, I detail mechanistic investigations of how GSK690693 and Y-33075 disrupt O-GlcNAc homeostasis. I find that these compounds have unique effects on OGT and OGA transcripts compared to previously characterized O-GlcNAc perturbogens. We further demonstrate that these compounds act orthogonally to previously characterized cis-regulatory elements within OGT. After identifying an alternative 5’ splice site induced by Y-33075 within OGA, we conducted RNA sequencing and related alternative splicing analyses, finding that GSK690693 is a broad inhibitor of intron processing and that Y-33075 is a specific modulator of exon inclusion across the transcriptome. I conclude with a discussion of the implications of these findings for therapeutic targeting of O-GlcNAc and other targets by splicing modulators and understanding cross-talk between O-GlcNAc and splicing.application/pdfenAlternative splicingChemical biologyGlycobiologyHigh-throughput screeningSmall molecule splicing modulatorChemistryBiochemistryThesis or Dissertation2026-01-130000-0002-0313-1538