Examining the role of Novel Thienopyridines as anti-cancer agents

Broughton, Christopher Paul (2024) Examining the role of Novel Thienopyridines as anti-cancer agents. Doctoral thesis (PhD), Manchester Metropolitan University.

Preview

Available under License Creative Commons Attribution Non-commercial No Derivatives. Download (16MB) | Preview

Abstract

Despite extensive research and significant advances, particularly over the past two decades, the treatment of mature B-cell malignancies remains a challenge. Drug resistance and disease relapse continue to occur and B-cell malignancies such as Chronic Lymphocytic Leukaemia (CLL), Follicular Lymphoma (FL) and Mantle Cell Lymphoma (MCL) largely remain incurable. This thesis focused on the application of a group of agents known as Thienopyridines in the treatment of mature B-cell malignancies. These novel molecules were developed by virtual screening followed by chemical synthesis at the University of Auckland, New Zealand. The ten compounds tested in this thesis are classed by their chemical structure into Cyclooctanes, Cycloheptanes and Cyclohexanones. The first experimental chapter explored the overall mode of effect of these compounds on the mature B-cell cell line, DAUDI, by identifying cytostatic and cytotoxic properties of the different compounds and relating the mode of action to the chemical structures. The effect of each of these compounds on the cell cycle was also investigated. After identifying that these compounds were inducing cell cycle arrest in the G2/M phase and inducing apoptosis, the second experimental chapter hypothesised, and demonstrated, that the G2/M arrest could be caused by effects on Tubulin polymerisation during spindle formation. The effect of these compounds on genes and proteins involved in (1) regulation of the Mitotic Checkpoint Complex (MCC) and Spindle Assembly Checkpoint, (2) mediators of the G2/M cell cycle checkpoint, and also (3) components of the B cell receptor signalling cascade was then specifically tested. The thesis was able to identify a group of the original ten Thienopyridines that show significant anti-neoplastic effects against the DAUDI cell line, mediated via tubulin destabilisation/stabilisation and activation of the SAC. As an aside, prior to any work investigating the effects of these compounds on gene regulation in this cell model, it was important to establish a robust set of reference genes for data normalisation. Chapter 5 reports on a systematic review of the literature that revealed a distinct lack of robust published data concerning reference genes in B-cell models and therefore went on to design primers for a panel of candidate reference genes and test these candidates in the experimental conditions for which they are required, thereby providing new data on a set of stable reference genes for use in RT-qPCR data normalisation. Overall, the data presented here define the Thienopyridines as a novel class of compounds that induce B-cell cytostasis or apoptosis via perturbation of microtubule polymerization and could be further developed as a new class of drug for the treatment of mature B-cell malignancies.