The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses. Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases. Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.

No description available.

No description available.

No description available.

DNA is both the oldest and newest of targets for cancer therapy. While it is already being targeted by many anticancer drugs in the clinic, the development of sequence-specific DNA binders has brought it back to the limelight as a valuable anticancer drug target.My studies on DNA interacting agents was initiated with the DNA intercalator campotothecin, and also included topoisomerase I enzyme. I have evaluated the structure of topoisomerase I C-terminal domain that consists of the active site tyrosine. My data indicate that this domain exists in a molten globule conformation with a fluctuating tertiary structure. These fluctuations are suggested to be important in interaction with the topoisomerase I core domain and DNA. I have also evaluated the DNA interactions of the camptothecin analogue homocamptothecin and have determined that homocamptothecin intercalate with DNA in the absence of topoisomerase I, and that such intercalation results in its lactone stabilization. Subsequently, the mechanism of topoisomerase I mediated inhibition of HIF-1 by camptothecin was explored. I have shown that camptothecin stimulate topoisomerase I cleavage complex formation in the HIF-1 binding site, which is suggested to prevent the DNA binding of HIF-1.The second part of this study was focused on understanding the mechanism of action of another DNA binder, XR5944. Designed as a dual topoisomerase inhibitor, XR5944 was subsequently shown to have a different mechanism of action - inhibition of trancription. The NMR structural analysis, in our lab, of the drug-DNA complex showed that XR5944 bis-intercalate with DNA, while binding in the DNA major groove. Driven by these combined interaction modes, XR5944 is shown to inhibit the DNA binding and the subsequent transcriptional activity of specific transcription factors such as estrogen receptors and AP-1, which are overexpressed in certain cancers.Finally, I have analyzed G-quadruplex structures formed by telomeric DNA. The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase. Thus the telomeric DNA G-quadruplex has been considered as an attractive anticancer drug target. Telomeric DNA forms multiple G-quadruplex conformations, and my data reveal the conformations of the major G-quadruplexes formed by human telomeres.

The anticancer properties of dirhodium tetraacetate were discovered in the 1970's, and subsequently motivated the research of several dirhodium paddlewheel derivatives. The promising results of this research led the Dunbar group to investigate the biological properties of dirhodium partial paddlewheel compounds. Previous work in our group has focused on dirhodium carboxylate derivatives with a series of diimine ligands, namely 1,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2a:2',3'c] phenazine (dppz), and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (dppn). Current research has expanded this diimine series by substituting the carobxylate bridging group with p-methoxyphenylphosphine (PMP). This new series of compounds was characterized by several techniques, including: X-Ray crystallography, 1H NMR spectroscopy, and electronic absorption spectroscopy. The cytotoxicity of these compounds towards HeLa cells was investigated in presence and absence of light in an effort to investigate the ability to use these compounds as photodynamic therapy (PDT) agents. Cytotoxicity measurements were carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. It was found that in the dark [Rh2(PMP)2(dppz)2][BF4]2 (the dppz derivative of the dirhodium PMP compound) had no cytotoxicity towards HeLa cells, but experienced a 7 fold increase in cytotoxicity upon irradiation (with lambdai_rr equal to 350 nm). This dramatic increase in cytotoxicity upon irradiation makes this compound a potential PDT agent. Diiridium (II,II) compounds were prepared in a dual attempt to determine how the properties of the dirhodium core effect the biological activities of these compounds, as well as investigate the biological activity of a set of compounds that has yet to be explored. The compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was chosen because it has a well understood dirhodium analogue, and it is a known compound. However, it was discovered that there was a potential silver contamination in the final product, stemming from the silver trifluoroacetate oxidant used during synthesis. Consequently, a new method of preparing this compound was required. The new synthetic pathway for the diiridium compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was devised, and the cytotoxicity and photocytotoxicity studies were performed for the first time (to our knowledge) on a diiridium (II,II) compound. Despite the stability of the compound, it was determined to be highly toxic, both in the dark and upon irradiation.

First isolated from the myxobacterium Sorangium cellulosm So Ce12 in 1994, the chivosazoles have been reported to possess antiproliferative activity against human cancer cell lines, as well as antifungal activity. This thesis focuses on studies towards the total synthesis of chivosazole F. Some developments towards the total synthesis of chivosazole A are also discussed. Chapter 1 discusses the isolation, characterisation and biological activity of the chivosazoles, as well as the first total synthesis of chivosazole F reported by the Kalesse group and the previous work towards synthesising chivosazole F in our group. Chapter 2 describes the synthesis of the three key fragments A, B and C, their coupling reactions and subsequent modifications for assembling the backbone of chivosazole F. Paterson boron aldol methodology and Evans-Tishchenko reduction were utilised to construct the 1,4-syn and 1,3-anti stereochemical relationships within both fragment A and fragment B. Di-tert-butyl silyl group was used for the efficient and precise protection of the terminal diol of B. The key stereochemistry of fragment C was defined with a vinylogous Mukaiyama aldol reaction. Site-selective Stille cross-coupling reactions of the three fragments, via a one-pot process, rapidly installed the requisite stereodefined polyene motifs within chivosazole F. Optimised Still-Gennari-type HWE olefination conditions were applied to install the (2Z,4E)-dienoate in D. MnO2-mediated double oxidation of D turned the terminal alcohol into an aldehyde and the oxazoline into an oxazole, followed by a Stork-Zhao olefination transforming the aldehyde to a Z-vinyl iodide for a macro-Stille coupling reaction, which achieved the ring closure to afford macrocycle E. Chapter 3 discusses the developments towards the synthesis of the southern fragment F of chivosazole A. Sugar I was prepared first and conditions were screened for the glycosylation of H and I to afford G. Chapter 4 outlines the achievements of this research and points out some future issues that need to be tackled.

Taxol®, an important anticancer drug, was first isolated in extremely low yield from the bark of the western yew, Taxus brevifolia. The clinical utility of Taxol has prompted a tremendous effort to obtain this complex molecule synthetically. Due to the chemical complexity of Taxol, its commercial production by total synthesis is not likely to be economical. Another natural product, 10-deacetyl baccatin III, is readily available in higher yield. Several methods have been reported for the synthesis of Taxol by coupling baccatin III and the N-benzoyl-β-phenylisoserine side chain. A new method for the synthesis ofTaxol from baccatin III is reported, and this method is compared with other methods. / Master of Science

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

Doctor of Philosophy(PhD) / The present study deals with the synthesis and characterization of six tri-palladium complexes code named MH3, MH4, MH5, MH6, MH7 and MH8 that contained two planaramine ligands bound to the central or each of the terminal metal ions. The activity of the compounds against human cancer cell lines: A2780, A2780cisR and A2780ZD0473R, cell uptake, levels of DNA-binding and nature of interaction with salmon sperm and pBR322 plasmid DNA have also been determined. Whereas cisplatin binds with DNA forming mainly intrastrand GG adduct that causes local bending of a DNA strand, the tri-palladium complexes are expected to bind with DNA forming a number of long-range interstrand GG adducts that would cause a global change in DNA conformation. Among the designed complexes, MH6 that has two 2-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be most active. The compound also has the highest cell uptake and Pd-DNA binding levels. In contrast, MH8 which has two 4-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be least active. The results indicate that, as applied to the terminal metal centres, 2-hydroxypyridine would be more activating than 4-hydroxypyridine perhaps because of greater protection provided to the terminal centres from coming in contact with the solvent molecules. In contrast, when bound to the central metal centre, 4-hydroxypyridine appears to play a slightly greater activating role than 2-hydroxypyridine or 3-hydroxypyridine, suggesting that non-covalent interactions such as hydrogen bonding associated with the ligand rather than its steric effect may be a more important determinant of antitumour property. The results illustrate structure-activity relationships and suggest that the tri-palladium complex containing two 2-hydroxypyridine ligands bound to each of the three metal centres or the compound that contains two 2-hydroxypyridine ligands bound to each of the two terminal metal centres and two 4-hydroxypyridine ligands bound to the central metal centre, may be much more active than any of the designed complexes.