Parmi les nombreux agents thérapeutiques utilisés en oncologie, le paclitaxel (Taxol®) est sans doute celui qui a suscité le plus d'intérêt. Il est utilisé en clinique pour le traitement des cancers de l'ovaire, du sein et des poumons. Il agit comme poison du fuseau mitotique en favorisant l'assemblage de la tubuline en microtubules et en stabilisant le polymère formé. Initialement extrait de l'if du Pacifique (Taxus Brevifolia) puis obtenu par hémisynthèse à partir de la 10-déacétylbaccatine III, il est aujourd'hui produit par un procédé biotechnologique de fermentation de cellules végétales. Le paclitaxel possède une structure chimique complexe basée sur un squelette tétracyclique taxane. Une approche visant à remplacer ce squelette taxane par une structure chimique plus simple a été entreprise afin d'identifier des mimes du paclitaxel. L'identification d'un fragment chimique (fragment based drug design) dérivé de la proline par une étude de modélisation moléculaire a permis de développer de nouvelles séries de mimes du paclitaxel. Parallèlement, le remplacement du squelette taxane par une matrice peptidique cyclique utilisant des dérivés de la proline a été réalisé. Les études de modélisation moléculaire, la synthèse des mimes du paclitaxel et leur évaluation biologique seront présentées. / Among the many therapeutic agents used in oncology, paclitaxel (Taxol®) is probably the one that generated the most interest. It is used clinically for the treatment of ovarian, breast and lung cancers and acts as a mitotic spindle poison by promoting the assembly of tubulin into microtubules and stabilizing the polymer formed. Initially extracted from the Pacific yew (Taxus Brevifolia) and obtained by semi-synthesis from 10-deacetylbaccatin III, it is now produced by a biotechnological process of cell plant fermentation. Paclitaxel has a complex chemical structure based on a tetracyclic taxane skeleton. A process to replace the taxane skeleton with a simpler chemical structure was undertaken to identify paclitaxel mimics. The identification of a chemical fragment (fragment based drug design) derived from proline by a molecular modeling study has led to the design of a new series of paclitaxel mimics. Meanwhile, replacing the taxane skeleton by a cyclic peptide scaffold using proline derivatives was performed. Molecular modeling studies, synthesis of paclitaxel mimics and biological evaluation will be presented.

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.

Indiana University-Purdue University Indianapolis (IUPUI) / The purpose of this prospective, observational study was to determine if a technique used to detect early signs of small-fiber neuropathy (local skin heating) could detect signs of small-fiber taxane-induced peripheral neuropathy (TIPN) in breast cancer survivors (BCS) during the first 6 weeks of Taxol®. Aims of the study were to compare the mean size of (1) axon reflexes and (2) axon flares (both markers of small fiber nerve function) in BCS receiving Taxol® to the size of reflexes/flares in healthy female controls (HCs). A third aim was to determine whether the size of axon reflexes/flares correlated with (a) overall TIPN severity and (b) severity of individual signs/symptoms of TIPN during early Taxol®. Data for the study was collected from nine BCS and 20 HCs (N = 29). All BCS had first-time, non-metastatic cancer and received weekly or bi-weekly Taxol®. Data was collected at 3 time-points: Time 1 (day 0, before Taxol®), Time 2 (day 14), and Time 3 (day 42). Axon reflexes and flares were generated using a validated 40-minute skin heating protocol. Axon reflexes were measured using laser Doppler Flowmetry. Axon flares were measured using full-field laser perfusion imaging. TIPN was measured using the 5-item Short Form of the Total Neuropathy Score (Reduced Version). Results identified potential signs of small-fiber TIPN in BCS after 6 weeks of Taxol®. Contrary to expectation, axon reflexes were larger for BCS at Time 3 than HCs, suggesting that Taxol® may be associated with an increase in small-fiber nerve function like that seen in pre-clinical studies. Clinical signs/symptoms of TIPN were not significantly correlated with axon reflexes or axon flares at the same time point. Analyses of axon flare size were confounded by issues with the data. These results add to the growing body of evidence showing that Taxol® affects small-diameter sensory nerves and provides the first evidence in humans that changes in small-fiber nerve function may be detectable after just 6 weeks of Taxol® therapy. Studies in larger samples are needed to validate these findings.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

No description available.

Selective killing of tumor cells requires the identification of drug targets critical to pathways that drive or support cancer progression. Protein kinases are an important class of intracellular enzymes involved in the regulation of biochemical pathways, deregulation of these kinases has been strongly implicated in cancer progression. To identify possible oncogenic kinases to which tumor cells might be selectively addicted, we screened the ON108 series of ATP-mimetic small molecule inhibitors in various triple negative breast cancer (TNBC) and normal cell lines. This approach led us to the identification of a novel kinase inhibitor, ON108600. We first examined the in vitro and in vivo effects of ON108600. ON108600 was found to be a potent inhibitor of Casein Kinase 2 (CK2) and the Dual-Specificity-Tyrosine (Y)-Phosphorylation-Regulated-Kinase (DYRK) family of serine-threonine kinases, both of which have been implicated in cancer progression. ON108600 showed broad-spectrum anti-proliferative and cytotoxic activity in multiple TNBC cell lines whilst having little or no effect on normal cells. Treatment of cancer cells with ON108600 resulted in inhibition of downstream signaling mediated by substrates of CK2. Further, ON108600 selectively arrested cancer cells in the mitotic phase of the cell cycle and activated the caspase-signaling cascade. We next performed x-ray crystallographic studies of ON108600-CK2 to determine the structural basis of ON108600-CK2 interaction. The co-crystal structure of ON108600-CK2 revealed that ON108600 binds in the active site pocket of CK2α wherein it mimics the binding of ATP and GTP in the CK2 active site. Notably, ON108600 mimics not only the shape and electrostatics of ATP/GTP, but also their hydration patterns in the CK2 active site pocket. Structural studies further revealed that ON108600 induces a conformational change in the β4-β5 loop of the catalytic subunit, which is known to interact with the β-regulatory subunit of CK2 and is critical for substrate recognition and activation. Lastly, we examined the efficacy of ON108600 in Triple Negative Breast Cancer (TNBC) and its ability to target and eliminate chemo-resistant Tumor-Initiating Stem Cells (TI-SCs) in TNBC. Clonogenic survival and sphere forming ability of purified CD44high CD24-/low TI-SCs from MDAMB-231 and Hs578t cells was potently inhibited by ON108600 treatment. We also observed that paclitaxel-resistant MDAMB-231 cells had increased levels of the CD44high CD24-/low stem cell- like population that correlated with increased expression of kinases CK2α2, DYRK1A and DYRK1B and these cells were sensitive to ON108600 treatment. Significantly, ON108600 showed robust antitumor efficacy as a single agent in a highly aggressive orthotopic TNBC xenograft model showing ~60% tumor growth inhibition. Immunohistochemical analysis of ON108600 treated tumors showed that a significant percentage of cells were apoptotic, indicating that activation of caspase mediated apoptosis contributes to the mechanism of action of ON108600 in vivo. Taken together, our results demonstrate that ON108600 is a novel and potent inhibitor of the CK2α1, CK2α2, DYRK1A and DYRK1B kinases. ON108600 binds in the active site pocket of CK2α and mimics ATP-GTP binding. ON108600 inhibits CK2-mediated signaling; arrests cancer cells in mitosis and induces apoptotic cell death via activation of caspases. Importantly, ON108600 is able to effectively kill the CD44high CD24-/low breast-cancer stem cell like population from TNBC cells. Finally, taxol-resistant MDAMB-231 TNBC cells express high levels of CD44, CK2α2, DYRK1a and DYRK1b and are sensitive to ON108600 treatment. Our study represents the first attempt to associate protein kinase CK2, DYRK1A and DYRK1B with TNBC and TI-SCs in TNBC and identifies a novel kinase inhibitor, ON108600 which effectively kills TI-SCs and taxol-resistant cells in TNBC. / Molecular Biology and Genetics

The complex natural product paclitaxel (Taxol®), first isolated from Taxus brevifolia, is a member of a large family of taxane diterpenoids. Paclitaxel is extensively used for the treatment of solid tumors, particularly those of the breasts and ovaries. In order to obtain additional information about the mechanism of action of paclitaxel and the environment of the paclitaxel-binding site, several fluorescent analogs of paclitaxel were synthesized, and their biological activities have been evaluated. For the investigation of possible synergistic effects, concurrent modifications on selected positions have been performed and their biological evaluation were studied. / Ph. D.

Prostate cancer is the most common non-skin cancer for men in America. The androgen receptor exerts transcriptional activity and plays an important role for the proliferation of prostate cancer cells. Androgen receptor ligands bind the androgen receptor and inhibit its transcriptional activity effectively. However, prostate cancer can progress to hormone refractory prostate cancer (HRPC) to avoid this effect. Chemotherapies are currently the primary treatments for HRPC. Unfortunately, none of the available chemotherapies are curative. Among them, paclitaxel and docetaxel are two of the most effective drugs for HRPC. More importantly, docetaxel is the only form of chemotherapy known to prolong survival in the HRPC patients. We hypothesized that the conjugation of paclitaxel or docetaxel with an androgen receptor ligand will overcome the resistance mechanism of HRPC. Eleven conjugates were designed, synthesized and biologically evaluated. Some of them were active against androgen-independent prostate cancer, but they were all less active than paclitaxel and docetaxel. Discodermolide is a microtubule interactive agent, and has a similar mechanism of action to paclitaxel. Interestingly, discodermolide is active against paclitaxel-resistant cancer cells and can synergize with paclitaxel, which make it an attractive anticancer drug candidate. Understanding the bioactive conformation of discodermolide is important for drug development, but this task is difficult due to the linear and flexible structure of discodermolide. Indirect evidence for the orientation of discodermolide in the tubulin binding pocket can be obtained from fluorescence spectroscopy of the discodermolide tubulin complex. For this purpose, we designed and synthesized a simplified fluorescently labeled discodermolide analog, and it was active in the tubulin assembly bioassay. In addition, a conformationally constrained discodermolide was designed to mimic the bioactive conformation according to computational modeling. The synthetic effort was made, but failed during one of the final steps. / Ph. D.

The conformation of paclitaxel in the bound state on the protein has been proposed to be the T-taxol conformation, and paclitaxel analogs constrained to the T-taxol conformation proved to be significantly more active than paclitaxel in both cytotoxicity and tubulin polymerization assays, thus validating the T-taxol conformation as the tubulin-binding conformation. In this work, eight compounds containing an aza-tricyclic moiety as a mimic of the baccatin core of paclitaxel have been designed and synthesized as water-soluble simplified paclitaxel analogs, among which 3.50-3.52 and 3.55 were conformationally constrained analogs designed to bind to the paclitaxel binding site of tubulin, based on their similarity to the T-taxol conformation. The open-chain analogs 3.41-3.43 and 3.57 and the bridged analogs 3.50-3.52 and 3.55 were evaluated for their antiproliferative activities against the A2780 cell lines. Analogs 3.50-3.52 and 3.55 which were designed to adopt the T-taxol conformation showed similar antiproliferative activities compared to their open-chain counterparts. They were all much less active than paclitaxel. In the second project, a series of paclitaxel analogs with various thio-containing linkers at C-2′ and C-7 positions were designed and synthesized in our lab. These analogs were attached to the surfaces of gold nanoparticles by CytImmune Sciences for the development of mutifunctional tumor-targeting agents. The native analogs and the gold bound analogs were evaluated for their antiproliferative activities against the A2780 cell line. All the compounds tested showed comparable or better activities than paclitaxel. Stability studies were performed for selected analogs in hydrolysis buffer, which showed that the analogs released paclitaxel in buffer over time. In the third project, the synthesis of a conformationally constrained paclitaxel analog which was designed to mimic the REDOR-taxol conformation was attempted. Two synthetic routes were tried and significant progress was made toward the synthesis of the conformationally constrained analog. However, both of the current synthetic routes failed to produce the key intermediate that would serve as the precursor for a ring-closing metathesis reaction to furnish the macrocyclic ring. / Ph. D.

Yes / Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer. / Cancer Research UK Project Grant (C16929/A14402) and the Elizabeth Blackwell Institute, through its Wellcome Trust ISSF Award (105612/Z/14/Z). The Imperial NIHR/Biomedical Research Centre and the CR-UK/Dept of Health funded Imperial Experimental Cancer Medicine Centre.