This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

Combinatorial protein libraries based on a protein template offer a vast potential for deriving protein variants harboring new receptor specificity while retaining other tem-plate functions to serve as library search-engines, cell-routing sequences and therapeutic domains. This concept was tested with the design and synthesis of protein libraries where short random peptide motifs were embedded directly within the catalytic A subunit of the bacterial ribosome-inactivating protein (RIP) known as Shiga-like toxin 1 (SLT-1). More precisely, a seven amino acid peptide epitope (PDTRPAP) was inserted between residues 245-246 of its A subunit (SLT-1APDTRPAP) and shown to preserve catalytic function while exposing the epitope. SLT-1 A chain libraries harboring tripep-tide and heptapeptide random elements were subsequently constructed, screened and shown to express more than 90% of expected cytotoxic A chain variants. Finally, more than 9,000 purified SLT-1 A chain variants were screened using their ribosome-inactivating function in a cell-based assay to identify mutants that are able to kill human melanoma 518-A2 cells. This search led to the striking discovery of a single chain RIP that displays selectivity for a panel of human melanoma cell lines as well as minimal immunogenicity when injected repeatedly into mice. This directed evolution of a RIP template provides a broad platform for identifying cell type specific cytotoxic agents.

Water soluble platinum complexes are a recent area of emphasis of cisplatin chemistry. The water soluble complexes could have a reduced toxicity compared with cisplatin. Oxaliplatin, which has an oxalate leaving group, has previously been shown to have less nephro-toxicity and higher water solubility than cisplatin. [Pt(en)(oxalate)] (en = ethylenediamine) has been prepared from Pt(en)Cl2 and silver oxalate. This complex has been reacted with methionine and N-acetylmethionine at different molar ratios. At high Pt: methionine ratios, chelates with the sulfur and nitrogen atoms of the methionine are dominant; at lower Pt: methionine ratios, a bis-methionine product is formed. The en ligand is displaced by methionine but not N-acetylmethionine.

One of the main problems of current cancer chemotherapy is the lack of selectivity of anti-cancer drugs to tumor cells which leads to systemic toxicity and adverse side effects. In order to overcome these limitations, researches on controlled drug delivery systems have gained much attention. Nanoscale based drug delivery systems provide tumor targeting. Among many types of nanocarriers, superparamagnetic nanoparticles with their biocompatible polymer coatings can be targeted to an intented site by an external magnetic field. Thus, the drug can be carried to the targeted site safely. The aim of this study is to prepare poly(dl-lactic-co-glycolic acid) (PLGA) coated magnetic nanoparticles and load anti-cancer drug, doxorubicin to them. For this purpose, magnetite (Fe3O4) iron oxide nanoparticles were synthesized as a magnetic core material (MNP) and then coated with oleic acid. Oleic acid coated MNP (OA-MNP) was encapsulated into PLGA. Effects of different OA-MNP/PLGA ratios on magnetite entrapment efficiency were investigated. Doxorubicin loaded magnetic polymeric nanoparticles (DOX-PLGA-MNP) were prepared. After the characterization of prepared nanoparticles, their cytotoxic effects on MCF-7 cell line were studied. PLGA coated magnetic nanoparticles (PLGA-MNP) had a proper size and superparamagnetic character. The highest magnetite entrapment efficiency of PLGA-MNP was estimated as 63 % at 1:8 ratio. Cytotoxicity studies of PLGA-MNP did not indicate any notable cell death between the concentration ranges of 2 and 250 &mu / g ml-1. It was observed that DOX-PLGA-MNP showed significant cytotoxicity on MCF-7 cells compared to PLGA-MNP. The results showed that prepared nanoparticles have desired size and superparamagnetic characteristics without serious toxic effects on cells. These nanoparticles may be suitable for targeted drug delivery applications. The findings obtained from drug studies may contribute to further work.

This dissertation research was aimed at investigating an interesting class of 1,2-dialkynylimidazoles as: 1. irreversible p38 MAP kinase α-isoform (p38α) inhibitors; 2. p38α docking site probes; 3. anti-cancer agents. Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles was designed as potential irreversible p38α inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activity were reported. Interestingly, one of the 1-ethynyl-substituted dialkynylimidazoles is a potent (IC50 = 200 nM) and selective inhibitor of p38α. Additionally, this compound covalently modifies p38α as determined by ESI-MS after 12 h incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by this compound demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors. Blocking docking interactions between kinase network partners is a promising alternative approach for selectively inhibiting kinases. The second project involves the identification of a new class of small molecules, covalent p38α MAP kinase docking site probes. We proposed that the mechanism may involve the addition of a thiol to the N-ethynyl group. Moreover, we demonstrated that such probes can be used fluorescently to label p38α both in vitro and in cells via azide-alkyne “Click” chemistry. This serves as the basis of an assay that can be used to identify inhibitors that specifically target the substrate docking site of p38α. The last project was focused on evaluating a new class of 1,2-dialkynylimidazoles as anti-cancer agents. One 1,2-dialkynylimidazole analog was found to be cytotoxic against a range of human cancer lines and to induce apoptosis in the human non-small cell lung cancer cell line A549. In order to elucidate the relationship between the structural basis and role of the thermal generation of diradical or carbene intermediates, a series of dialkynylimidazoles and related N-alkynylimidazoles was prepared and their cytotoxicity was determined against A549 cell line. Although the experimentally determined activation energy is in excellent agreement with that predicated from the DFT calculation, there is no correlation between the rate of Bergman cyclization and cytotoxicity to A549 cells. An alternative mechanism was proposed involving the unexpected selective thiol addition to the N-ethynyl group of certain 1,2-dialkynylimidazoles. / text

Novel therapies and delivery methods directed against malignancies such as melanoma, and particularly metastatic melanoma, are needed. The HIV-1 accessory protein Vpr (viral protein R) has previously been demonstrated to induce G2 cell cycle arrest as well as in vitro growth inhibition/killing of numerous tumor cell lines. In vivo electroporation has been utilized as an effective delivery method for pharmacologic agents as well as DNA plasmids that express "therapeutic" proteins and has been targeted to various tissues including malignant tumors. In this study, we assessed the ability of electroporation-mediated delivery of Vpr plasmid (pVpr) to induce growth attenuation or complete tumor regression in C57BL/6 mice with subcutaneous B16.F10 melanoma lesions. To assess the administration of intratumoral delivery of pVpr with in vivo electroporation, a range of Vpr plasmid dosages, electroporation parameters, and treatment days were evaluated in a subcutaneous B16 murine melanoma model. pVpr was injected directly into the tumors. Immediately following the injection, the subcutaneous tumors were electroporated. Treatment with 25 microgram or 100 microgram of pVpr plus electroporation on days 0 and 4 resulted in complete tumor regressions with long-term survival in 14.3% and 7.1% of the mice, respectively. In order to optimize the treatment regimen, B16 tumors were treated on days 0, 2, and 4 with 100 microgram pVpr plus electroporation which resulted in 50% of the mice with complete tumor regressions and long-term survival. Additional investigations revealed intratumoral Vpr expression and demonstrated that apoptosis was the mechanism by which Vpr caused tumor regression in vivo. This study confirmed that treatment with 100 microgram of pVpr plus electroporation led to durable complete regressions in established murine melanoma lesions. The pVpr plus electroporation treatment regimen has induced complete regressions in mice as well as resistance to tumor challenge in some of the animals. This is the first comprehensive study demonstrating the ability of Vpr, when delivered as a DNA expression plasmid with in vivo electroporation, to induce complete tumor regressions coupled with long- term survival of mice in a highly aggressive and metastatic solid tumor model.

Four analogues of the Lewis X trisaccharide antigen (β-D-Galp(1→4)[α-L-Fucp(1→3)]-D-GlcNAcp) in which the galactosyl residue is modified at O-4 as a methyloxy, deoxychloro, deoxyfluoro or deoxy were synthesized. The preparation of the modified 4-OMe, 4-Cl, 4-F and 4-H trichloroacetimidate galactosyl donors is described as well as their use in the glycosylation of an N-acetylglucosamine glycosyl acceptor. The resulting disaccharides were deprotected at O-3 of the glucosamine residue and fucosylated giving access to the desired protected Lewis X analogues. One step global deprotection (Na/NH3) of the protected 4”-methoxy and 4”-deoxy analogues, and two step deprotections (removal of a p-methoxybenzyl with DDQ, then Zemplén deacylation) of the 4”-deoxychloro and 4”-deoxyfluoro protected Lex analogues gave the desired compounds in good yields. The relative binding energies of the 4” manipulated Lewis X methyl glycoside analogues for anti-Lewis X mAb SH1 were determined using competitive ELISA procedures. The binding experiments revealed that substitution of the 4” site with a deoxychloro, deoxyfluoro or deoxy function had a large differential change in the free energy of binding; yet they did not completely abolish cross reactivity with native Lewis X. Substitution with a methyloxy function eliminated recognition by anti-Lewis X monoclonal antibody SH1. / NSERC

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.