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Epidemiological and preclinical studies suggest that dietary-derived cancer chemopreventive agents may be active at dietary and supra-dietary doses. This project therefore investigated the effect of dose on the pharmacokinetic and pharmacodynamic actions of resveratrol, a polyphenol found in grapes. An achievable dietary intake (5 mg/day in humans) and a supra-dietary dose, previously used in clinical trials, were compared in preclinical systems, in healthy volunteers and in patients. Accelerator mass spectrometry (AMS) was used in the clinical part of this project as its sensitivity allowed the safe administration of radiolabelled resveratrol to participants. In vitro microarray results from human colon HCA7 cancer cells showed that resveratrol exposure for 3 months at 0.01 μM, the estimated plasma level in man after an oral 5 mg dose, altered the apoptosis, glucose transport and cell adhesion pathways. Resveratrol exposure at 1.4 μM, the plasma Cmax value in man after a 1 g dose, did not significantly alter any pathways. The equivalent doses, determined by conversion by body weight and resulting in the same tissue levels as in man, were administered to ApcMin+/- mice. Resveratrol reduced the tumour burden at a dietary dose in the presence of a high fat diet, but this was not seen when administered with a standard fat diet, nor at supra-dietary doses. The mechanisms of actions of resveratrol in vivo were unclear. Clinically, oral administration of [14C]-resveratrol resulted in detectable levels in plasma, colon and prostate tissue even at the 5 mg dose. The metabolite profiles were similar between prostate tissue and plasma, with metabolites predominating. Overall, these results suggest that the dose-response relationship of resveratrol may be biphasic or U-shaped. The confirmation of detectable levels of resveratrol and its metabolite in prostate and colon tissue suggest that resveratrol could exert pharmacodynamic actions in these organs.

The eukaryotic translation initiation factor 4E is an indispensable component of the cap-dependent translation initiation process. eIF4E binds to the mRNA cap and recruits, with the rest of initiation factors, the mRNA to the ribosomes. Under normal physiological conditions, eIF4E is the least abundant element of the translation initiation machinery. However, it has become evident that eIF4E is overexpressed in numerous tumour types leading to a translation enhancement of the oncogenic mRNAs. Thus it contributes to the initiation as well as the progression of the disease. Although eIF4E has been validated as a cancer drug target for over two decades, no drug-like eIF4E inhibitor has been developed, apart from the antiviral drug ribavirin. In this project, a set of nucleotide-based eIF4E inhibitors was designed and synthesised to mimic the mRNA cap structure in order to block the eIF4E cap binding slot. These molecules have a modified m7GTP structure at the level of the phosphate moiety as well as the N7 substituents, sites that are directly linked to the ligand affinity for eIF4E. A series of competitive and functional assays was then developed and optimised with the aim of assessing the ability of these ligands to sequester eIF4E from binding to capped mRNAs. These assays examined the inhibitors at multiple levels including the binding to a recombinant eIF4E, the effect on eIF4E functional role and the cellular influence on protein synthesis.

Mammalian cells are able to transduce signals via protein pathways, and this enables cells to grow, differentiate and survive in a controlled fashion. This regulation is often lost in cancer, leading to the development of malignant tumours. Cytotoxic therapy such as radiotherapy and chemotherapy are commonly used to treat cancer. Numerous pharmacological drugs have also recently been developed, in an attempt to inhibit the action of these oncogenic proteins and to prevent their aberrant action, or to sensitise cancer cells to cytotoxic therapy. However, little is known about the actions of these inhibitors on the tumour microenvironment, which amongst other components, is comprised of blood vessels. Tumour vasculature is poorly organised, dilated, irregular in distribution and shape, and is less mature than its normal counterpart. The failure of cancer chemotherapy is partly thought to be due to the failure of cytotoxic drug delivery to the tumour. Since effective drug penetration relies in part on an effective blood supply throughout the tumour, the ability to modulate the tumour microenvironment is one possible strategy to help achieve better drug distribution. The inefficient vasculature also results in tumour hypoxia. Clinically, hypoxic tumours are more resistant to radiotherapy, in part because oxygen is required for the maximal fixation of radical species which can cause DNA damage. Increasing oxygen levels within tumours may thus improve the efficiency of radiotherapy. Here, we characterise vascular changes in the tumour microenvironment upon signalling inhibition. After the restriction of oncogenic signalling, tumour vasculature becomes more structurally normal with improved flow, which leads to a corresponding reduction in tumour hypoxia. These changes result in improved chemotherapy drug delivery and the enhancement of radiotherapy which, in turn delays tumour growth. We demonstrate that modulation of the tumour microenvironment in this way is a viable strategy to improve the effectiveness of cytotoxic therapy.

Nosocomial and community-acquired infections due to methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria are associated with high levels of mortality, morbidity and significant social and economic costs in the U.K. and elsewhere. As the evolution of multi-drug resistance relentlessly erodes the utility of currently available antibacterial drugs, it is essential to maintain efforts to search for new classes of antibacterial agents. Benzimidazoles are potent antihelmintic agents discovered in 1961. Chemical modifications led to the synthesis of the head-to-tail fluorochrome Hoechst 33258 and, more recently, to symmetric head-to-head bis-benzimidazoles (sBBZ). The antibacterial potential of these compounds was examined. Antibacterial activity was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays using a total of 143 Gram-positive isolates, including 61 MRSA, 12 vancomycin resistant enterococci (VRE), 12 Streptococcus pneumoniae, 11 Listeria monocytogenes, 7 Mycobacterium spp. (including Mycobacterium tuberculosis) and 14 Gram-negative isolates. Structure-activity relationships were determined and key requirements for activity were identified. Acute toxicity of sBBZs was assessed using the sulforhodamine B staining method and a zebrafish embryo model. To clarify mode of action, time kill studies, flow cytometry, DNA microarrays and radioisotope incorporation assays were employed. sBBZs displayed activity against Gram-positive but not Gram-negative pathogens; the most potent compound possessed MIC90 values of 0.06, 0.125 and 1 mg/L against, respectively, MRSA, VRE and M tuberculosis isolates. Data suggests that sBBZs are bacteriostatic agents that interfere primarily with the DNA machinery and do not select for drug resistant variants over a 31-day period of drug exposure. Subtle structural modifications have an incisive effect on in vitro potency of sBBZs. Toxicity was determined as minimal for WI-38 cell line and the zebrafish embryo model of infection. Sub-inhibitory concentrations of sBBZs inhibited MRSA transcription of pathogenicity-associated genes. Symmetric bis-benzimidazoles are new DNA-interfering bacteriostatic agents with potent antibacterial activity and significant therapeutic potential against Gram-positive bacteria, including MRSA and M tuberculosis.

The Chorioallantoic Membrane (CAM) assay can be used to assess a treatment's anti-angiogenic potential; but also, it can function as a tumour model in its own right. Tumourigenic cells are implanted in the embryo early in development, with these cells present before the development of the immune system, they are determined to be 'self. From here, the tumour cells divide, usurp a blood supply for nutrients and oxygen and begin to form a tumour mass. This mass can then be subjected to treatments and can determine a treatment's anti-angiogenic or anti-tumour efficacy. The recruitment of a blood supply, the process of angiogenesis, without which tumour cells cannot replicate beyond a defined volume as they are confined by the laws of diffusion. Zoledronate (ZA) has previously been shown to possess some anti-angiogenic properties. ZA is used clinically to treat cancer associated bone disease and, as a fourth generation bisphosphonate, is very effective at preventing skeletal remodelling. The target of nitrogen containing bisphosphonates, such as ZA, is the mevalonate pathway; this pathway is responsible for the generation of lipids used to anchor proteins to the plasma membrane. With fewer proteins anchored in the plasma membrane, pro-survival signalling pathways are adversely affected. The CAM assay was established and refined; multiple cancer cell lines and some human tumour tissue were engrafted, so displaying intriguing histological architecture. Real time biomarkers of tumour growth were also investigated. Examination of the molecular changes required for adaption from in vitro to tumour models revealed several interesting avenues of research, which are currently being pursued. The use of the CAM model in the evaluation of oncolytic viruses was also investigated, with results suggesting a model worthy of further investigation. ZA was shown to be cytotoxic to endothelial cells and induced apoptosis with little effect on the cell cycle. Treatment of endothelial cells with Vascular Endothelium Growth Factor (VEGF) increased their resistance to ZA-induced cytotoxicity. The concentration of ZA required to inhibit the growth, or induce cytotoxicity, in a panel of renal cancer cell lines were much greater than that of endothelial cells. Apoptosis and. Caspase assays, as well as cell cycle analysis, varied in result between renal cancer cell lines. VEGF had no effect on the proliferation rate of these cancer cell lines and an appropriate in vivo dose of a vascular disruptor was proposed. Neither ZA nor VEGF had any effect on the rate of tumour growth, either in the CAM or murine flank models. Only the vascular disruptor managed to impede tumour growth, but only for a time. Overall, the CAM assay is a useful technique and further investigations will elucidate some of the molecular mechanisms behind it. ZA has a modest anti- angiogenic capability, but, its inability to hamper tumour growth, compared to that of an established drug, indicates further experimentation and analysis is required

PTEN and LKB 1 are linked tumour suppressor genes that regulate cell polarization, a key event of tissue morphogenesis. The small Rho GTPase, Cdc42 is responsive to PTEN, LKBI and is the ultimate effector of polarized growth, mediated through the PAR6/aPKCζ polarization complex. This thesis investigates dialogue between PTEN, LKBI and Cdc42-dependent two- or three-dimensional (2D or 3D) polarized cell growth. Signalling interactions and 2D polarized growth were investigated in monolayer cultures of PTEN expressing or deficient isogenic or non-isogenic cells, complemented by studies in LKB 1 constitutively deficient LKB 1 cells. Relevant molecular pathways were investigated by transfection and/or pharmacological studies. PTEN and LKBI enhance Cdc42 activity. PTEN is a negative regulator of PI3- kinase (pI3K) signaling while LKBI activates AMPK. In separate investigations of these downstream pathways, we found that PTEN influences Cdc42 activity. We also found that aPKCζ mediated Cdc42 activation via LKB 1. Furthermore, LKB 1 - AMPK targeted therapy substantially enhanced Cdc42 activity. Both PTEN and LKB 1 enhance Cdc42-dependent 2D polarization, shown by assays of reorientation of the microtubule organizing centre (MTOC), monolayer wound assays and AMPK targeted therapy can rescue the MTOC defect of PT EN deficiency. In 3D culture, PTEN expressing Cac02 cells formed regular gland like structure with a single central lumen. Conversely, PTEN deficient Cac02 ShPTEN cultures had an abnormal multi lumen or multivacuolar phenotype that could be rescued by PTEN targeted therapy. Additionally, treatment of Caco2 ShPTEN cultures with APMK targeted therapy rescued morphogenesis, restoring a single central lumen. Taken together, these studies show dialogue between PTEN and LKB 1 pathways in regulation of cdc42-dependent polarization signaling. This principle can be exploited for rescue of defective 2D polarized growth and 3D morphogenesis.

Pyrimidine biosynthesis represents an attractive target for chemotherapeutic intervention in the treatment of a number of diseases. The enzyme dihydroorotate dehydrogenase (DHODH), which catalyses the fourth reaction in the de novo pyrimidine .biosvnthesis pathway, has for many years received particular attention as a drug target. Human DHODH is the target of the antirheumatic drug leflunomide, and other inhibitors of this enzyme, such as brequinar, have been extensively investigated for their anticancer properties. Furthermore, the malaria parasite Plasmodium falciparum lacks the ability to salvage pyrimidines, meaning that the de novo synthesis pathway represents a potential target for the development of novel anttmalarials. Recent studies have shown that inhibitors of P. falciparum DHODH are able to suppress parasite growth in mice, validating this approach as a means of combating malaria. In this study, X-ray crystallography has been used to rationalise the inhibition patterns observed in two series of compounds that inhibit human and P. falciparum DHODHs, as well as aiding the design of inhibitors with increased potency. The best compounds from a set of human DHODH inhibitors were co-crystallised with their target enzyme, and were found to induce a small conformational change in the N-terminal helix of the enzyme that has not been observed previously. A new crystal form of PfDHODH has been discovered, leading to the determination of a 'number of eo-crystal structures at higher resolution than has been achieved by other groups. These structures reveal a subtle change in the rotamer adopted by a key arginine residue, allowing these compounds to form an additional hydrogen bond to the enzyme, relative to what has previously been reported with similar inhibitors. Virtual high-throughput screening has also been used to discover novel inhibitors of human and P. falciparum DHODHs. Biochemical assays showed a number of naphthoquinone-based compounds to be inhibitors of DHODH, and in silico docking has been used to try and explain their activity.

In 2007 over 12 million people were diagnosed with cancer. At least one third of these individuals are not expected to survive the disease, making cancer the second most prevalent cause of death worldwide. Systemic chemotherapy forms the mainstay of cancer treatment. In particular, this thesis focuses on antimitotic drugs, which are now common chemotherapeutic agents used to treat a wide variety of cancers. These compounds have seen success in the clinic, however, patient response remains highly unpredictable and resistance to treatment is commonplace. Anti-mitotic drugs such as taxol and the vinca alkaloids are thought to inhibit tumour proliferation by disrupting formation of the mitotic spindle, preventing normal Prolonged mitotic arrest due to chronic activation of the spindle assembly checkpoint (5AC). It is presumed that this prolonged arrest leads to cell death, however, the molecular links between mitotic arrest and cell death are ill-defined.

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