University of Macau / Institute of Chinese Medical Sciences

Le cancer de l’ovaire est la cinquième cause de décès par cancer chez la femme en France. La prolifération tumorale dans ce cancer est liée principalement à la dérégulation de l’apoptose. Cette dérégulation joue un rôle clé principalement dans la pathogénèse et la progression tumorale mais aussi dans le développement des résistances aux chimiothérapies existantes. La croissance accrue des cellules tumorales dans le cas du cancer de l’ovaire est liée à une surexpression des protéinesanti-apoptotiques de la famille Bcl-2, en particulier les protéines MCL-1 et Bcl-xL. L’inhibition concomitante de ces deux protéines conduit à la mort des cellules chimiorésistances. A ce jour, seuls les inhibiteurs de Bcl-xL ont démontré une efficacité en clinique, et l’inhibition de MCL-1 reste problématique dans un contexte clinique. Dans ce contexte, l’objectif principal de ces travaux de thèse consiste à caractériser, par RMN et modélisation moléculaire, l’interaction entre la protéine MCL-1 et le Pyridoclax, foldamère oligopyridinique très prometteur, synthétisé au CERMN. La spectroscopie RMN nous a permis de définir le site d’interaction, d’estimer la constante d’affinité et de guider le Docking pour obtenir une structure tridimensionnelle du complexe MCL-1:Pyridoclax. Afin de conforter notre étude sur l’implication du Pyridoclax dans l’interaction, des fragments dérivés de ce dernier ont été utilisés. La caractérisation de l’interaction entre ces fragments et la protéine MCL-1 a permis de relever l’importance du groupement styryle du Pyridoclax dans l’interaction avec la protéine MCL-1. Par ailleurs, nous avons élargi notre étude à d’autres inhibiteurs potentiels de MCL-1 tels que certains médicaments existant sur le marché. La spectroscopie RMN a permis de mettre en évidence et de caractériser l’interaction entre deux de ces médicaments (le Torsémide et le Déférasirox) et la protéine MCL-1. Pour finir, afin d’améliorer l’affinité de ces médicaments pour la protéine MCL-1, des modifications structurales ont été proposés en se basant sur la littérature. Les dérivés proposés semblent avoir une affinité très importante pour la protéine MCL-1. / Ovarian cancer is the fifth leading cause of cancer death of women in France. Tumor proliferation in this cancer is mainly related to the deregulation of apoptosis. This one plays a main role in pathogenesis and tumor progression but also on the term of development of resistance to existing chemotherapies. The increased growth of tumor cells in ovarian cancer is linked to an overexpression of the anti-apoptotic proteins of the Bcl-2 family, in particular the MCL-1 and Bcl-xL proteins. The concomitant inhibition of these two proteins leads to the death of chemoresistance cells. To date, only Bcl-xL inhibitors have demonstrated clinical efficacy andinhibition of MCL-1 remains problematic in a clinical context. In this context, the aim of this thesis is to characterize, by NMR and molecular modeling, the interaction between MCL-1 protein and potential inhibitors including Pyridoclax, an oligopyridine foldamer. NMR spectroscopy allowed us to define the interaction site, estimate the affinity constant and guide Docking to obtain a three-dimensional structure of the MCL-1:Pyridoclax complex. In order to obtain more information on the involvement of Pyridoclax in this interaction, fragmentsderived from it has been used. The characterization of the interaction between these fragments and MCL-1 protein revealed the importance of the styryl group of Pyridoclax in the interaction with MCL-1 protein. Furthermore, we expanded the scope of our study to include some existing drugs able to interact with MCL-1. NMR spectroscopy allowed us to identify and characterize the interaction between two of these drugs (Torsemide and Deferasirox) and the MCL-1 protein. Finally, in order to improve the affinity of these drugs for MCL-1 protein, structural modifications had been proposed based on the literature. The proposed derivatives appear to have a very high affinity for MCL-1 protein.

Ongoing research in the fields of genetics and biotechnology hold the promise of improved diagnosis and treatment of genetic diseases, and potentially the development of individually tailored pharmaceuticals and gene therapies. Difficulty, however, arises in determining how these services are to be evaluated and integrated equitably into public health care systems such as Canada's. The current context is one of increasing fiscal restraint on the part of governments, limited financial resources being dedicated to health care, and rising costs for new health care services and technologies. This has led to increasing public debate in the last few years about how to reform public health care, and whether we should prohibit, permit or perhaps even encourage private purchase of health care services. In Canada, some of these concerns have crystallized around the issue of gene patents and commercial genetic testing, in particular as illustrated by the case of Myriad Genetics' patented BRACAnalysis test for hereditary breast and ovarian cancer. While most Canadians who currently access genetic services do so through the public health care system, for those with the means, private purchase is becoming an option. This situation raises serious concerns - about justice in access to health care; about continued access to safe and reliable genetic testing supported by unbiased patient information; and about the broader effects of commercialization for ongoing research and the Canadian public health care system. Commercial genetic testing presents a challenge to health care professionals, policy analysts, and academics concerned with the social, ethical and policy implications of new genetic technologies. Using the Myriad case as an exemplar, tools from moral philosophy, the social sciences, and health policy and law will be brought to bear on the larger issues of how as a society we should regulate commercial research and product development, and more coherently decide which services to cover under public health insurance and which to leave to private purchase. Generally, the thesis is concerned with the question of "how best to bring capital, morality, and knowledge into a productive and ethical relationship" (Rabinow 1999, 20).

Ongoing research in the fields of genetics and biotechnology hold the promise of improved diagnosis and treatment of genetic diseases, and potentially the development of individually tailored pharmaceuticals and gene therapies. Difficulty, however, arises in determining how these services are to be evaluated and integrated equitably into public health care systems such as Canada's. The current context is one of increasing fiscal restraint on the part of governments, limited financial resources being dedicated to health care, and rising costs for new health care services and technologies. This has led to increasing public debate in the last few years about how to reform public health care, and whether we should prohibit, permit or perhaps even encourage private purchase of health care services. In Canada, some of these concerns have crystallized around the issue of gene patents and commercial genetic testing, in particular as illustrated by the case of Myriad Genetics' patented BRACAnalysis test for hereditary breast and ovarian cancer. While most Canadians who currently access genetic services do so through the public health care system, for those with the means, private purchase is becoming an option. This situation raises serious concerns - about justice in access to health care; about continued access to safe and reliable genetic testing supported by unbiased patient information; and about the broader effects of commercialization for ongoing research and the Canadian public health care system. Commercial genetic testing presents a challenge to health care professionals, policy analysts, and academics concerned with the social, ethical and policy implications of new genetic technologies. Using the Myriad case as an exemplar, tools from moral philosophy, the social sciences, and health policy and law will be brought to bear on the larger issues of how as a society we should regulate commercial research and product development, and more coherently decide which services to cover under public health insurance and which to leave to private purchase. Generally, the thesis is concerned with the question of "how best to bring capital, morality, and knowledge into a productive and ethical relationship" (Rabinow 1999, 20). / Graduate and Postdoctoral Studies / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.

Indiana University-Purdue University Indianapolis (IUPUI) / All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.