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The field of cancer therapeutic development has been dominated by two research and discovery paradigms, the cytotoxicity-based or phenotype driven strategy and the target-based rational approach. This thesis describes the standardization of novel assays used in both approaches and the discoveries made using these processes. Rational drug design or the target-based approach to discovering novel anti-cancer agents requires a basic understanding of the oncogenic signals that induce uncontrolled cellular proliferation. c-MET is a proto-oncogene, linked to a number of different cancers, that encodes a receptor tyrosine kinase. As an oncogene, c-MET has been shown to transform cells in the laboratory setting and is dysregulated in number of malignancies. Thus, we sought to discover a small molecule inhibitor of c-MET kinase activity by screening a novel library of small molecules. In the second part of this dissertation, we describe the standardization of a high-throughput assay to identify putative c-MET inhibitors and the results of our screening attempt. Cytotoxicity-based screening is another validated approach that is used to discover anti-cancer agents. As a parallel program to our c-MET discovery effort, we designed a high-throughput cytotoxicity assay to identify a novel small molecule with high cytotoxic activity towards tumor cells. The result of this screen was the identification of ON015640, a novel anti-cancer therapeutic with tubulin-depolymerizing activity. Throughout the course of this project, we tried to discern the advantages and disadvantages of the two predominant paradigms in cancer therapeutic research. Both strategies require careful assay design and an acute understanding of the molecular and genetic underpinnings of cancer. While it is clear that structure-based rational drug design has its merits and its success stories, it has become increasingly clear that seeking out a desired biological effect may serve as a more effective staring point when dealing with cancers for which no clear oncogene addiction phenotype has been observed. / Molecular and Cellular Physiology

Yes / There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

Despite recent advancements in cancer treatment, the management of heterogeneous cancers poses a persistent challenge. This is best exemplified by the limited success in treating acute myeloid leukemia (AML), which still exhibits a 5-year survival rate of less than 30% to date. The resistance to treatment and high likelihood of relapse in AML patients is believed to be attributed to the elusive nature of cancer stem cells (CSCs). CSCs are characterized by their self-renewal capabilities and blockade to normal differentiation into mature hematopoietic cells, however they are only functionally defined through their engraftment potential upon transplantation into immunodeficient mice to phenocopy human tumorigenesis. Therefore isolation, study and treatment of CSCs is a prominent challenge in cancer research. To circumvent this challenge, our research group has developed a proprietary screening platform capable of identifying compounds within vast chemical space that selectively target surrogate human CSCs while preserving healthy stem cell counterparts. Our approach of screening and identifying compounds that selectively target human CSCs compared to normal SCs focuses on custom compound libraries curated through pre-screening for anti-proliferative and lethality properties in Escherichia coli (E. coli). From a pool of 21,000 active molecules selected from several libraries chemical space totalling 8.7 million compounds, we identified 15 distinct structural frameworks or "scaffolds", each representing different chemical structures that form the basis for potential drug development. Through secondary assays using primary AML patient derived cells, we have prioritized a novel compound, termed MLMB-2231 as a promising lead anti-CSC candidate. However, beyond MLMB-2231's demonstrated ability to selectively target human CSCs over normal stem cells, the cellular and molecular mechanisms of activity are unknown, limiting improvements and use towards investigational new drug application (IND) initiation. Here, I have utilized a variety of chemical genomics techniques to probe the downstream effects and gene targets of MLMB-2231. Apoptosis and cell cycling assays demonstrated that MLMB-2231 operates through an induction of apoptosis at 48h resulting in a G0/G1 cell cycle stall. The use of genome-wide CRISPR viability screening combined with transcriptomic analysis through RNA sequencing identified significant upregulation of pathways associated with aberrant ubiquitin- protease system (UPS), disruption of cell cycling, and upregulation of apoptotic pathways. These findings suggest that MLMB-2231 exerts its effects by interfering with the UPS, leading to impaired protein degradation and cellular stress, disrupting key cell cycle checkpoints, and inducing apoptosis. Future studies will focus on hit validation to confirm direct binding targets and incorporate animal models to evaluate pharmacokinetics (pK) and overall efficacy in vivo, paving the way for potential clinical applications. / Thesis / Master of Science (MSc)

Yes / The integrin family of cell surface extracellular matrix binding proteins are key to several physiological processes involved in tissue development, as well as cancer proliferation and dissemination. They are therefore attractive targets for drug discovery with cancer and non-cancer applications. We have developed a new integrin antagonist chemotype incorporating a functionalised cyclobutane ring as the central scaffold in an arginine–glycine–aspartic acid mimetic structure. Here, we report the synthesis of cyclobutanecarboxylic acids and cyclobutylamines with tetrahydronaphthyridine and aminopyridine arginine mimetic sidechains and masked carboxylic acid aspartic acid mimetic sidechains of varying length. Effective αvβ3 antagonists and new aspartic acid mimetics were identified in cell-based adhesion and invasion assays. A lead compound selected based on in vitro activity (IC50 < 1 μM), stability (t1/2 > 80 minutes) and synthetic tractability was well-tolerated in vivo. These results show the promise of this synthetic approach for developing αvβ3 antagonists and provide a firm foundation to progress into advanced preclinical evaluation prior to progression towards the clinic. Additionally, they highlight the use of functionalised cyclobutanes as metabolically stable core structures and a straightforward and robust method for their synthesis. This important contribution to the medicinal chemists' toolbox paves the way for increased use of cyclobutanes in drug discovery. / This work was funded by Yorkshire Cancer Research (Award reference number B002-PhD) and Prostate Cancer UK (Pilot Grant PA10-01).

Thesis (LL.M.). / Written for the Institute of Comparative Law. Title from title page of PDF (viewed 2008/01/14). Includes bibliographical references.

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