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.

This project applies structure-activity relationship (SAR), structure-based and database mining approaches to study ligand-protein interactions. To support these studies, we have developed a relational database system called EDinburgh University Ligand Selection System (EDULISS 2.0) which stores the structure-data files of +5.5 million commercially available small molecules (+4.0 million are recognised as unique) and over 1,500 various calculated molecular properties (descriptors) for each compound. A user-friendly web-based interface for EDULISS 2.0 has been established and is available at http://eduliss.bch.ed.ac.uk/. We have utilised PubChem bioassay data from an NMR based screen assay for a human FKBP12 protein (PubChem AID: 608). A prediction model using a Logistic Regression approach was constructed to relate the assay result with a series of molecular descriptors. The model reveals 38 descriptors which are found to be good predictors. These are mainly 3D-based descriptors, however, the presence of some predictive functional groups is also found to give a positive contribution to the binding interaction. The application of a neural network technique called Self Organising Maps (SOMs) succeeded in visualising the similarity of the PubChem compounds based on the 38 descriptors and clustering the 36 % of active compounds (16 out of 44) in a cluster and discriminating them from 95 % of inactive compounds. We have developed a molecular descriptor called the Atomic Characteristic Distance (ACD) to profile the distribution of specified atom types in a compound. ACD has been implemented as a pharmacophore searching tool within EDULISS 2.0. A structure-based screen succeeded in finding inhibitors for pyruvate kinase and the ligand-protein complexes have been successfully crystallised. This study also discusses the interaction of metal-binding sites in metalloproteins. We developed a database system and web-based interface to store and apply geometrical information of these metal sites. The programme is called MEtal Sites in Proteins at Edinburgh UniverSity (MESPEUS; http://eduliss.bch.ed.ac.uk/MESPEUS/). MESPEUS is an exceptionally versatile tool for the collation and abstraction of data on a wide range of structural questions. As an example we carried out a survey using this database indicating that the most common protein types which contain Mg-OATP-phosphate site are transferases and the most common pattern is linkage through the β- and γ-phosphate groups.