Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis.

Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis.

This thesis describes the development of a small molecule sensor for the detection of specific protein-protein interactions. The responsive sensor has been designed to produce a measurable signal upon a bivalent interaction with a dimeric protein target, and has the potential to probe the biological significance of specific binding events.

Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This thesis describes the development and refinement of a number of techniques for molecular docking and ligand database screening, as well as the application of these techniques to predict the structures of several protein-ligand complexes and to discover novel ligands of an important receptor protein. Global energy optimisation by Monte-Carlo minimisation in internal co-ordinates was used to predict bound conformations of eight protein-ligand complexes. Experimental X-ray crystallography structures became available after the predictions were made. Comparison with the X-ray structures showed that the docking procedure placed 30 to 70% of the ligand molecule correctly within 1.5A from the native structure. The discrimination potential for identification of high-affinity ligands was derived and optimised using a large set of available protein-ligand complex structures. A fast boundary-element solvation electrostatic calculation algorithm was implemented to evaluate the solvation component of the discrimination potential. An accelerated docking procedure utilising pre-calculated grid potentials was developed and tested. For 23 receptors and 63 ligands extracted from X-ray structures, the docking and discrimination protocol was capable of correct identification of the majority of native receptor-ligand couples. 51 complexes with known structures were predicted. 35 predictions were within 3A from the native structure, giving correct overall positioning of the ligand, and 26 were within 2A, reproducing a detailed picture of the receptor-ligand interaction. Docking and ligand discrimination potential evaluation was applied to screen the database of more than 150000 commercially available compounds for binding to the fibroblast growth factor receptor tyrosine kinase, the protein implicated in several pathological cell growth aberrations. As expected, a number of compounds selected by the screening protocol turned out to be known inhibitors of the tyrosine kinases. 49 putative novel ligands identified by the screening protocol were experimentally tested and five compounds have shown inhibition of phosphorylation activity of the kinase. These compounds can be used as leads for further drug development.

The failure of the pharmaceutical industry to increase the delivery of new drugs into the market is driving a re-assessment of practices and methods in drug discovery and development. In particular alternative strategies are being pursued to find therapeutics that are more selective, including small molecules that target protein-protein interactions. However, success depends on improving our understanding of the recognition of small molecules by interfaces in order to develop better methods for maximising their affinity and selectivity, whilst trying to confer an appropriate therapeutic profile. This thesis starts with the description of the creation of TIMBAL, a database that holds small molecules disrupting protein-protein interactions. The thesis then focuses on the analysis of these molecules and their interactions in a medicinal chemistry and structural biology context. TIMBAL molecules are profiled against other sets of molecules (drugs, drug-like and screening compounds) in terms of molecular properties. Using the structural databases in the Blundell group, the atomic detail of the interaction patterns of TIMBAL molecules with their protein targets are compared with other molecules interacting with proteins, comprising natural molecules, small peptides, synthetic small molecules (including drug-like and drugs) and other proteins. The structural features and composition of the binding sites of these complexes are also analysed. Keeping in mind that current drug candidates are somewhat too lipophilic to succeed, these interaction profiles are defined in terms of polar and apolar contacts, with the aim of migrating natural patterns into the design of new therapeutics.

No description available.

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)