Development of Innovative Chemical Tools for Studying O-GlcNAc Transferase Enzyme

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

O-GlcNAcylation can be defined as an addition of O-GlcNAc to the Serine/Threonine residue of proteins by the enzymatic activity of O-GlcNAc transferase (OGT) enzyme. O-GlcNAcylation is considered a dynamic and reversible process as another enzyme called O-GlcNAcase (OGA) undoes the action of OGT. OGT being one of the only two enzymes modulating O-GlcNAcylation, has a pivotal role in a wide range of biological processes such as cell signalling and communication, cell division and invasion, transcription and translation of factors/co-factors. The roles played by O-GlcNAcylation and OGT in the pathology of metabolic disorders such as cancer, diabetes and neurodegenerative diseases have been documented in detail over the last few decades. However, its exact molecular mechanism is poorly understood due to the lack of specific tools to perturb its functionality within the biological systems. Therefore, attempts have been made to construct specific chemical tools in the form of OGT inhibitors but the lack of potency and selectivity has slowed down their progress. In the current thesis, focus was set on the construction of novel, specific and potent inhibitors of the targeted OGT enzyme with a view to gain a better understanding of its active role. In addition, an attempt was made to investigate the impact of OGT inhibition on the cytotoxic effects exerted by the anticancer agents. Overall, the outcomes of this work further enhanced our understanding of the intricacies involved in the development of selective and potent OGT inhibitors. Chapter-1 presents the introduction of the OGT enzyme and helps to understand the significance of studying O-GlcNAcylation and the OGT enzyme in addition to outlining the major aims of the thesis. This chapter contains literature review paper specifically focusing on the role of O-GlcNAcylation and OGT enzyme in the stabilization of various oncogenic factors (manuscript-1) Chapter-2 describes the specific details on the materials and methodology used in chapter 3 to 5. Chapter-3 discusses the design and development of novel inhibitors targeting the OGT enzyme using bisubstrate analogues as a template. This strategy employed the identification of suitable donor and acceptor substrates where a flexible aliphatic linker was introduced to replace the negatively charged pyrophosphate group. This was based on the hypothesis that such exchange could improve the cell permeability of inhibitors. Uridine and GlcNAc were selected as donor substrate as UDP-GlcNAc is acting as a donor substrate for OGT enzyme for O-GlcNAcylation. The results demonstrated that one compound from the series, compound-17 (uridine-O-peptide conjugate, manuscript-2) could inhibit the human OGT enzyme with an IC50 value of 29.34 μM. In-silico prediction for physicochemical properties showed that compound-17 has higher value of Log P than previously reported bisubstrate inhibitors (i.e. Goblin-1 and Thiogoblin-1) which means theoretically compound-17 has better cell permeability. Chapter-4 describes another series of bisubstrate inhibitors where uridine-mimetic scaffolds were selected as donor substrate. In this chapter, attempt was made to understand the impact of an exchange of modifiable amino acid (serine) with unmodifiable amino acid (alanine) as well as D-serine to evaluate chirality. One of the candidates from that series of compounds (compound-29, manuscript-3) was also found to have moderate inhibitory activity against OGT with an IC50 value of 134.40 μM. These preliminary findings laid a foundation for the better understanding of the nuances required within the basic scaffold of bisubstrate analogue inhibitors to achieve desired selectivity and potency. Chapter-5 discusses the exploration of a combinatorial approach involving an OGT inhibitor (OSMI-1) with existing chemotherapeutic agents (doxorubicin and docetaxel). OGlcNAcylation and OGT are indispensable for the signal transduction and activation of various oncogenic factors that are thought to be responsible for the reduced sensitivity of prostate cancer cells towards these anticancer agents (manuscript-4). Investigation of concomitant treatment of doxorubicin with an OGT inhibitor demonstrated synergistic effects in terms of cell death in prostate cancer cells (manuscript-5). Chapter-6 presents concluding remarks on the outcomes of the proposed work and future directions.