Gastrointestinal malignancies such as liver and pancreatic cancers are the deadliest due to late detection and drug resistance. Nucleoside analogues, like Gemcitabine, are the conventional therapy despite their little impact on survival and off-target toxicity. A novel class of nucleoside analogues able to evade drug resistance mechanisms has been developed by the Guindon group and biologically screened in our lab. Some of these proprietary molecules were further equipped with a lipoate moiety designed to target cancer cell metabolism. LCB2151 and LCB2179 have emerged as the lead molecules in this class, with an IC50 of 10-15 µM in the Gemcitabine-resistant human pancreatic (Capan-2 & Panc-1) cancer cell lines. The focus of this project is deciphering the cellular mechanisms activated by LCB2151 in these pancreatic cancer lines. A series of biased molecular approaches, like gene expression profiling, and unbiased large throughput proteomic and metabolomics analyses were applied to identify potential targets and affected pathways. Results collectively show that LCB2151 evades drug resistance mechanisms, increases pro-apoptotic markers and impairs mitochondrial respiration as early as 6 hours posttreatment. Furthermore, MS/MS analyses reveal that LCB2151 alters the levels of several metabolites in the central carbon metabolism pathway and identifies the citric acid cycle enzyme α-ketoglutarate dehydrogenase as a potential molecular target of LCB2151. Understanding the exact mechanism of action of our lead molecule along with extensive testing on murine cancer models, will surely pave its way to clinical testing and evaluation.

Supramolecular polymers have a practical impact on the healthcare field as they can act as scaffolds to repair parts of organs such as the brain or heart. In addition, they can provide insight into theories relating to the origins of life. For instance, the hypothesis that RNA played a more important role in early biology, the RNA World hypothesis, would be strengthened if there were a way to show the spontaneous formation of RNA-like polymers from monomer units. However, the natural nucleobases do not assemble at the monomer level, nor do they form nucleosides readily with ribose, leading some to speculate that the first nucleobases may have been different from the ones used in biology today. This conundrum encouraged us to begin looking for alternative nucleobases that are able to self-assemble into polymers capable of storing information. Our lab has recently demonstrated that a modified 2,4,6-triaminopyrimidine (TAP) will assemble with cyanuric acid (CA) in water through interactions that are analogous to those between complementary nucleobases found in DNA and RNA. When TAP is modified at one of its three faces, it can pair through specific hydrogen bonding with CA on two of its faces, forming rosette structures. These rosettes self-assemble to form extremely long structures through the stacking of tens of thousands of rosettes. In this study we are investigating prebiotically relevant syntheses of TAP nucleosides. Using chromatography techniques and nuclear magnetic resonance we found that the unmodified TAP with D-ribose formed nucleosides in 60% yields with the major product (20%) being a C-nucleoside 5-β-ribofuranosyl-2,4,6-triaminoprymidine or TARC. TARC forms hydrogels with CA, both in the crude reaction and after purification, indicative of the formation of supramolecular polymers out of a complex mixture. The results of this study provide support for the possibility of pre-RNA molecules.

The immune-reactive sperm are kept separate from the body by epithelial barriers such as the blood-testis barrier (BTB). While these barriers are beneficial for the protection of sperm from toxicants, they can make treating these areas difficult due to preventing the entry of pharmacological agents. This is especially an issue in the treatment of HIV and Ebola infection based on the ample evidence that these viruses are able to survive and spread from within the male genital tract (MGT), but only a few antiviral drugs are known to access the MGT. Transporters that line the epithelial barriers of the MGT, especially the BTB, are important for determining whether or not a drug is able to penetrate into the MGT through transepithelial transport. Several nucleoside analogs (NSA), which are used to treat HIV infection and leukemias, are known to be able to accumulate in seminal plasma, which makes them a useful tool for understanding transepithelial transport for the BTB. The purpose of these studies is to characterize the transport profile for the MGT, in particular the BTB, to gain a better understanding of how xenobiotics, especially ones based on nucleosides, can access the MGT. The chief finding of this work is the discovery of a transepithelial transport pathway expressed by Sertoli cells that allows for the entry of nucleosides (necessary for germ cell development) and NSA into the MGT. This pathway depends on equilibrative nucleoside transporter (ENT) 1 uptake and ENT2 efflux and occurs in both rats and humans. These studies provide the foundation for being able to predict the penetration of novel drugs into the MGT.

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Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound. In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes. An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness. Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products.

Les traitements actuels contre le virus de l’hépatite B (VHB) combinent un ou plusieurs analogues de nucléos(t)ides qui inhibent directement la réplication virale en bloquant l’étape de transcription inverse. Ces traitements très efficaces sont pourtant confrontés à l’émergence de virus résistants à ces traitements. Ces résistances sont la conséquence de l’émergence et la sélection de mutants parfois complexes présentant des mutations à la fois dans le gène de la polymérase (pol) et de l’enveloppe virale. Les objectifs principaux de ce doctorat ont été d’étudier la sensibilité des variants résistants du VHB vis-à-vis d’analogues de nucléos(t)ides et de nouveaux composés nonnucléos(t)idiques agissant contre la nucléocapside, mais également de comparer les performances virales de différents mutants afin de comprendre le processus de sélection des mutants qui s’opère chez le patient sous pression thérapeutique. Ces études ont caractérisé la sensibilité de certaines mutations de résistance aux analogues de nucléos(t)ides, de souligner l’importance des modifications de l’enveloppe dues aux mutations de résistance dans le processus d’émergence et de sélection des variants dans la quasi-espèce virale et d’identifier de nouvelles molécules antivirales efficaces permettant, en combinaison avec les analogues de nucléos(t)ide, de diminuer fortement les phénomènes de résistance du VHB. Mieux comprendre les phénomènes de résistance, les procédés d’émergence, de sélection et de transmission des mutants du VHB pour élaborer les meilleures stratégies cliniques de combinaisons thérapeutiques peut réduire considérablement le nombre de personnes touchées par ce virus / Current therapies against the hepatitis B virus (HBV) combine one or more nucleoside analogues that directly inhibit viral replication by blocking reverse transcription step. These treatments are very effective, however, faced with the emergence of viruses resistant to these treatments. These resistances are the result of the emergence and selection of mutants with mutations can be complex in both the polymerase gene (pol) and the viral envelope. The main objectives of this PhD was to study the sensitivity of resistant HBV variants vis-à-vis similar nucleos(t)ides and new compounds non-nucleos(t)idic acting against the nucleocapsid, but also compare the performance of different viral mutants to understand the process of selection of mutants that occurs in patients under therapeutic pressure. These studies have characterized the sensitivity of some resistance mutations to nucleoside analogues, to highlight the importance of the envelope changes due to resistance mutations in the process of emergence and selection of variants in the quasispecies virus and to identify new effective antiviral drugs may allow, in combination with nucleoside analogues, to greatly reduce the phenomenon of HBV resistance. Better understanding the phenomenon of resistance, the processes of emergence, selection and transmission of HBV mutants to develop the best clinical strategies of combination therapy can significantly reduce the number of people affected by this virus

La prise en charge du cancer reste dépendante de l’utilisation des agents cytotoxiques, avec les analogues nucléosidiques. Au-delà de leur similarité structurelle, certains de ces composés partagent une voie métabolique commune, où la cytidine déaminase apparaît comme enzyme majeure. L’existence d’une variabilité génétique et/ou phénotypique de la CDA nous a mené à nous intéresser aux relations entre le statut CDA et l’issue clinique des patients afin de déterminer si la CDA pouvait être considérée comme biomarqueur d’issue clinique chez les patients.Nos travaux personnels ont consisté à évaluer deux techniques permettant de mesurer l’activité de la CDA. Nous avons publié le premier cas mondial de toxicités mortelles sous azacytidine chez un patient CDA-déficient, ainsi que le premier cas de déficience en CDA et de toxicités sous cytarabine causées par la présence d’une variation génétique du gène CDA chez une patiente transplantée hépatique. L’influence du statut CDA a également été étudiée chez deux patients traités par azacytidine. Concernant la gemcitabine, nous avons démontré l’impact délétère en terme d’efficacité de l’augmentation de l’activité CDA chez les patients, ainsi que les résultats d’une étude multicentrique prospective dont le but était de déterminer si la CDA pouvait être un marqueur prédictif de l’apparition des toxicités sous gemcitabine, avec une étude pharmacocinétique en support. Les travaux préliminaires du pyroséquençage partiel de la CDA sur technologie Roche® sont présentés. L’ensemble de ces travaux de thèse confirme l’intérêt d’évaluer le statut CDA chez les patients susceptibles de recevoir une thérapie à base d’analogues nucléosidiques. / Nowadays, the management of cancer pathology remains largely dependent on the use of cytotoxic agents, including nucleoside analogs, used in a variety of settings. Beyond their structural similarity, some of these compounds also share a common metabolic pathway, wherein the cytidine deaminase (CDA) plays a pivotal role. The existence of constitutional genetic and / or phenotypic variability in CDA prompted us to study the relationships between the CDA status and clinical outcome in patients, and to determine if the constitutional CDA could be considered as a biomarker of efficacy and toxicity in patients treated with this class of drugs.Our personal work first consisted in evaluating two methods to measure the CDA enzymatic activity, in terms of robustness and cost. Then we published the first case-report of life-threatening toxicities in a CDA-deficient patient treated with azacytidine, and the first case of CDA deficiency and cytarabine-caused toxicities correlated with presence of a genetic variation in CDA gene in a liver-transplant patient. The influence of CDA status was also assessed in two patients treated with azacytidine. Regarding gemcitabine, we present the impact of an increase in CDA activity on loss of efficacy in patients, and the results of a prospective multicenter study whose purpose was to determine whether the CDA could be a predictive marker of the occurrence of gemcitabine-toxicities, with a pharmacokinetic study support. Finally, preliminary data on partial Roche®-pyrosequencing of CDA, also presented.All these thesis work confirms the interest to evaluate the CDA status in patients likely to receive a nucleosidic analogues-based therapy.

No description available.

No description available.