Tankyrases-1 and -2 (TNKS-1 and -2) are members of the poly(ADP-ribose)polymerase (PARP) enzyme superfamily, which modify and regulate target proteins by addition of multiple (ADP-ribose) units from the substrate NAD+. TNKS-1 and -2 have many cellular roles, including regulation of elongation of telomeres, activation of nuclear mitotic apparatus protein (NuMA) in mitosis and regulation of the Wnt signalling pathway. This makes the tankyrases attractive new targets for design and development of new anti-cancer drugs. 2-(4-Trifluoromethylphenyl)-7,8-dihydro-3H-thiopyranopyrimidin-4-one (XAV939) was one of the few active inhibitors of tankyrases reported until 2013. The aim of this project was to explore the structure-activity relationships towards enhancing potency and selectivity by replacing the saturated sulfur-containing ring with saturated and unsaturated nitrogen heterocycles and by varying the aromatic side-chain. Firstly, ascorbate-modified Sonogashira couplings of bromocyanopyridines and a variety of 4-substituted arylethynes, followed by acidic cyclisation and conversion of the lactone into the lactam, gave differently substituted arylnaphthyridinones. The alternative route used transition-metal-free reaction of bromopyridinecarboxylic acids with symmetrical β-diketones. 7-Phenyl-1,6-naphthyridin-5-one and 7-(4-methylphenyl)-1,6-naphthyridin-5-one were converted to the N1-oxides. Alkylation at 1-N gave 7-aryl-1-methyl-5-oxo-5,6-dihydro-1,6-naphthyridin-1-ium iodides and subsequent reduction gave saturated target 7-aryl-1-methyl-1,2,3,4-tetrahydro-1,6-naphthyridin-5-ones. Other target compounds included pyridopyrimidinones, which were prepared from the corresponding bromopyridinecarboxylic acids by a copper-catalysed reaction with 4-substituted benzamidines. Target tetrahydropyridopyrimidinones, however, were obtained from condensation of 1-benzyl-4-oxopiperidine-3-carboxylic esters with substituted benzamidines. All compounds were evaluated in vitro for inhibition of the catalytic activity of TNKS-2. The best compounds were investigated further, including in vitro TNKS-1 and PARP-1 inhibition and anti-proliferative studies on HT29 and FEK4 cell lines. Notably, 1-methyl-7-(4-trifluoromethylphenyl)-1,2,3,4-tetrahydro-1,6-naphthyridin-5-one and 1-methyl-7-(4-methoxyphenyl)-1,2,3,4-tetrahydro-1,6-naphthyridin-5-one showed 50% inhibition of TNKS-2 at 1.5 nM and 1.1 nM, respectively, showing also high selectivity (IC50 against PARP-1: 4.8 μM and 3.4 μM, respectively). This high potency and selectivity point to potential for development towards therapeutic use in cancer. A patent covering these discoveries has been filed.

Poly(ADP-ribose) polymerase (PARP) represents a large family of enzymes that are activated upon DNA breakage, which are then involved in a cascade of reactions that eventually lead to cell death. PARPs, known to cause a variety of damage to the human body, are targets of many researches’ investigating potential inhibition mechanisms. To this point, plenty of PARP inhibitors have been synthesized and tested for potency against many diseases. Some have great potency against particular diseases, while others are already being used as drugs. The current research suggests a potential synthesis of novel PARP inhibitors, to be accomplished through Diels-Alder chemistry. The proposed compounds consist of an isoindolinone core bearing different functional groups. Three different strategies are described for the synthesis of the novel PARP inhibitors and all protocols involve sulfur dioxide extrusion chemistry to create a diene, the desired transient starting material. The diene is envisioned further to be reacted with a variety of dienophiles to give possibly potential PARP inhibitors. / NSERC

Les poly(ADP-ribose) polymérases (PARPs/ARTDs) sont des senseurs des cassures de l’ADN et utilisent la nicotinamide adénine dinucléotide (NAD+) pour synthétiser un polymère de poly(ADP-ribose) (PAR) long et branché attaché aux résidus accepteurs des protéines nucléaires. Le travail présent montre que les protéines des mammifères PARP-1, PARP-2 et PARP-3 sont capables d’ADPribosyler les extrémités des duplex d’oligonucléotides in vitro. PARP-1 modifie préférentiellement les substrats resequés, alors que PARP-2 et PARP-3 modifient préférentiellement les substrats avec un nick, et sont capables d’ADP-ribosyler les groupements phosphate en 5’ ou 3’ au niveau des extrémités des cassures double brin des duplex d’ADN contenant à proximité un nick ou gap phosphorylé en 5’. Cet étude présente une caractérisationdétaillée des préférences de substrat d’ADN pour les enzymes structuralement proches PARP-2 et PARP-3, et propose un modèle putatif du mécanisme de l’ADP-ribosylation des extrémités de l’ADN catalysée par PARP-3 et PARP-2. L’ADPribosylationefficiente d’un fragment d’ADN d’environ 3kb par PARP-3 et PARP-2, l’ADPribosylationmédiée par les PARPs dans les extrait cellulaires ainsi qu’un signal persistant générée par l’anticorps anti-PAR sur de l’ADN génomique purifié en série à partir des cellules HeLa déplétées de PARG et traitées à la bléomycine suggèrent que certains types de cassures complexes de l’ADN peuvent être efficacement PARylés par les PARPs comme réponse cellulaire aux dommages de l’ADN. Cet nouveau type de modification postréplicative de l’ADN médiée par les PARPs in vitro apporte des nouveaux éléments sur les mécanismes moléculaires sous-jacents aux enzymes qui catalysent l’ADP-ribosylation. / Poly(ADP-ribose) polymerases (PARPs/ARTDs) act as DNA break sensors and use nicotinamide adenine dinucleotide (NAD+) to catalyze the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor residues of nuclear proteins. The present work shows that mammalian PARP-1, PARP-2 and PARP-3 can ADP-ribosylate strand break termini in DNA oligonucleotide duplexes in vitro. PARP-1 preferentially modifies recessed substrates, while PARP2 and PARP3 preferentially modify nicked DNA and can ADPribosylate 5′- and 3′-terminal phosphate residues at double-strand break termini of a DNA duplex containing a proximal 5′-phosphorylated nick or a gap. This work provides detailed characterization of DNA substrate preferences for structurally

Les modifications post-traductionnelles des protéines de réparation de l’ADN et des facteurs chromatiniens par poly(ADP-ribose)ylation et par phosphorylation sont essentielles pour le maintien de l’intégrité de l’ADN et de la chromatine, en particulier dans la réponse cellulaire aux dommages de l’ADN induits par radiation ionisantes (RI). Parmi les protéines impliquées dans ces deux processus nous trouvons, respectivement, la poly(ADP-ribose) polymérase-1 (PARP-1) et PARP-2, et la kinase dépendante des cyclines Cdk5 : PARP-1 et PARP-2 sont impliqué dans le mécanisme de réparationdes cassures simples brin (CSBs) de l’ADN (Single Strand Break Repair : SSBR ) et la déplétion de Cdk5 a été liée à l’augmentation de la sensibilité des cellules aux inhibiteurs de PARPs. Nous avons montré, en utilisant des cellules HeLa stablement déplété pour Cdk5 ou PARP-2, que ces deux protéines sont impliquées dans les deux sous-voies du SSBR, le short- (SPR) et le long-patch repair (LPR). L’absence de Cdk5 ou PARP-2 entraîne des modifications du fonctionnement du SSBR, notamment en termes de recrutement des protéines de réparation PARP-1 et XRCC1, impliquées dans le SPR, et PCNA, protéine clé du LPR, au site du photo-dommage. PARP-2 et Cdk5 agissent aussi sur la balance du niveau des poly(ADP-ribose) car en absence de Cdk5 une hyper-activation de PARP-1 a été montrée, et en absence de PARP-2 une diminution de l’activité de la protéine poly(ADP-ribose) glycohydrolase (PARG) a été aussi observée. Cependant, malgré ces changements les deux lignées cellulaires dépourvues de Cdk5 (Cdk5KD) ou de PARP-2 (PARP-2KD) réparent de façon normale les CSBs radio-induites, mais, intéressement et contrairement aux cellules PARP-2KD, les cellules Cdk5KDsont sensibles à l’effet létal des RI. De plus nous avons montré que Cdk5, PARP-2 et PARG sont toutes les trois impliquées dans la régulation du recrutement et de dissociation du facteur chromatinien ALC1 suggérant leur implication dans la régulation de la dynamique de la chromatine en réponse aux photo-dommages de l’ADN. Ces résultats avec l’observation de la diminution du recrutement de PARP-1 dans les cellules Cdk5KD et PARP-2KD, montrent l’apparition d’un réseau complexe de phosphorylation et de poly(ADP-ribose)ylation en réponse aux RI qui implique Cdk5 , PARP-1,PARP-2 and PARG et qui est fort probablement initié par l’activité kinase de Cdk5. / The post-translational modifications of DNA repair proteins and histone remodeling factors by poly(ADP-ribose)ylation and phosphorylation are essential for the maintenance of DNA integrity and chromatin structure, and in particular in response to DNA damaging produced by ionizing radiation (IR). Amongst the proteins implicated in these two processes are the poly(ADP-ribose) polymerase -1 (PARP-1) and PARP-2, and the cyclin-dependent kinase Cdk5: PARP-1 and 2 are involved in DNA single strand break (SSB) repair (SSBR) and Cdk5 depletion has been linked with increased cell sensitivity to PARP inhibition. We have shown by using HeLa cells stably depleted for either CdK5 or PARP-2, that the recruitment profile of PARP-1 and XRCC-1, two proteins involved in the short-patch (SP) SSBR sub-pathway, to DNA damage sites is sub-maximal and that of PCNA, a protein involved in the long-patch (LP) repair pathway, is increased in the absence of Cdk5 and decreased in the absence of PARP-2 suggesting that both Cdk5 and PARP-2 are involved in both SSBR sub-pathways.PARP-2 and Cdk5 also impact on the poly(ADP-ribose) levels in cells as in the absence of Cdk5 a hyper-activation of PARP-1 was found and in the absence of PARP-2 a reduction in poly(ADP-ribose) glycohydrolase (PARG) activity was seen. However, in spite of these changes no impact on the repair of SSBs induced by IR was seen in either the Cdk5 or PARP-2 depleted cells (Cdk5KD or PARP-2KD cells) but, interestingly, increased radiation sensitivity in terms of cell killing was noted in the Cdk5 depleted cells. We also found that Cdk5, PARP-2 and PARG were all implicated in the regulation ofthe recruitment and the dissociation of the chromatin-remodeling factor ALC1 from DNA damage sites suggesting a role for these three proteins in changes in chromatin structure after DNA photodamage.These results, taken together with the observation that PARP-1 recruitment is sub-optimal in both Cdk5KD and PARP-2KD cells, show that an intricate network of phosphorylation and poly(ADPribose) ylation occurs in response to IR that involves Cdk5 , PARP-1,PARP-2 and PARG and that is probably initiated by Cdk5’s kinase activity.

Dans le cadre des recherches menées au sein du laboratoire de Physiologie Animale le gène codant pour la nicotinamide phosphorybosyltransférase (NAmPT) à été identifié et cloné. Au cours de ce travail, nous avons étudié le rôle de cette enzyme du métabolisme du Nicotinamide Adénine Dinucléotide ainsi que celui des enzymes dépendantes du NAD (PARP et sirtuines) dans le contrôle de la réponse inflammatoire.

Drug toxicity may cause liver injury, resulting in damage to cells and tissues. This damage can lead to cytotoxic events that may result in an activation of poly (ADP-ribose) polymerase (PARP). A study was conducted to determine if cocaine and acetaminophen toxicity lead to DNA damage and to the activation of the repair protein, PARP in the liver using the hepatotoxicants: cocaine and acetaminophen (APAP). A dose-response analysis for cocaine concluded that a dose as low as 20 mg/kg resulted in elevated ALT levels. A higher dose of 60 mg/kg was tested for analyses but resulted in severe hemorrhaging. The dose-response analyses for APAP resulted in no elevated liver enzyme levels for a 75 mg/kg and 150 mg/kg dose. A dose of 50 mg/kg for cocaine, and a dose of 300 mg/kg for APAP were used to analyze temporal trends for both toxicants. Both cocaine and APAP produced incremental increases in ALT at the 2 hour, 6 hour, 18 hour, and 24 hour time points, respectively. PARP activity analysis for cocaine measured the highest activity at the 2hr and 6hr time points. PARP analysis for acetaminophen measured gradual increases until the 18 hour time point where the highest level of PARP activity was measured. A PARP inhibition analysis was conducted with cocaine and (APAP) to understand the impact of a PARP inhibitor, 1,5-dihydroxyisoquinoline (DIQ), on PARP activity in the liver. A 50 mg/kg dose of cocaine or a 300 mg/kg dose of APAP was administered, followed by a 10 mg/kg dose of DIQ at 1) the time of initial toxicant dose (0 hour), or 2) 1 hour after initial toxicant dose (1hr). The PARP inhibition analysis for cocaine and APAP was conducted at 6 and 18 hours post initial dose, respectively, when the highest levels of PARP were observed. Inhibition analyses determined that ALT declined significantly when DIQ was administered immediately following the initial toxicant dose for both toxicants. DIQ administered 1 hr after initial toxicant dose resulted in slightly higher ALT than the 0 hr time point. Decreases in PARP activity were observed at the 0 hr time point, with slightly higher PARP levels observed at the 1 hr time point. Decreased PARP activity was observed following DIQ treatment with both, a concurrent drug treatment and treatment following drug administration. Cocaine and APAP treatment did not cause DNA fragmentation. A liver glutathione (GSH) analysis conducted for cocaine and APAP did not correlate with DIQ alteration of PARP activity. The mechanism of DIQ effects on drug-induced hepatotoxicity appears to be GSH independent. DIQ was effective in reducing drug-induced hepatotoxicity and preserving organ function.

Flow chemistry can be characterized as a continuous chemical reaction system performed in solution in connecting tubing and flow reactors which is efficient. Photochemistry is the chemical reaction initiated by light, and is the result of the absorption of photon by a reagent or starting material. Poly (ADP-ribose) polymerase is a big family of proteins related to cellular repair and death. Phenanthridinones have been shown to exhibit PARP inhibitory potency as competitive inhibitors. Instead of using conventional costly and low-efficiency coupling reactions, we have managed to develop a method to synthesize phenanthridinone-type compounds by photo-cyclization under flow conditions for the purposes of generating novel PARP inhibitors. In total, we have generated a series of phenanthridinones in yields ranging from 13 % to 99%, 18 examples. Additionally, we have also developed a flow photocyclization method for the synthesis of complex heterocycles, naphthyridinones (5 examples, yields ranging from 24-52%) and thieno-quinolinones (18 examples, yields ranging from 23-90%), molecules that would be much more difficult to construct using conventional batch methods. Overall, we have demonstrated that a flow photocyclization pathway is a robust synthesis route for producing phenanthridinone-type compounds for the purposes of developing novel PARP inhibitors. / October 2016

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

Prostate cancer is one of the most malignant tumors in the world. It has been demonstrated that prostate cancer could metastasis to bones, bladder, rectum, and lymph nodes. It is the most common type of cancer found in adult males, and the mortality is elevated. From screening our chemical library of 398 small molecule compounds, we have found that TCH derivatives showed anti-proliferative activities using prostate cancer cell line PC-3. The results of MTT assay allow us to identify TCH-1038w as potential candidat for developing anticancer drug. Morphological investigation on PC-3 cells after TCH-1038w treatment showed that PC-3 cells rounded up and combined with cell shrinkage, abridge, membrane blebbing. Our results of flowcytometric analysis showed that TCH-1038w can cause the percentage increase of sub-G1 that indicated the DNA fragmention in TCH treated PC-3 cells. By DAPI staining , we observed that TCH-1038w can induce the DNA fragmentation in PC-3 cells. Moreover by immunoblotting analysis, we have demonstrated that procaspase 3 and PARP were cleavaged and activated after TCH treatment. These results indicsted TCH drugs can induce caspase activation, DNA fragmentation, and consequently cause apoptotic cell death. Together, TCH-1038w may serve as a potential chemotherapy candidate for treating prosate cancer in the future.

The maintenance and regulation of cellular NAD(P)(H) content and its influence on cell function involves in many metabolic pathways which are poorly understood. Niacin deficiency in humans, which leads to low NAD status, causes sun sensitivity in skin, indicative of DNA repair deficiencies. Animal models of niacin deficiency demonstrate genomic instability and increased cancer development in sensitive tissues including skin. Therefore, we have developed a cell culture model that allows assessment of pathways regulated by NAD(P) content as a way to identify NAD-dependent signaling events that may be critical in early skin carcinogenesis. Using our model, we showed that niacin restriction, and consequent NAD depletion, reversibly alters NAD(P)(H) pools, increases apoptosis, induces G2/M cell cycle arrest, and decreases DNA stability. These alterations are affected by increased expression and activity of NOX leading to an accumulation of ROS, which may provide a survival mechanism as has been shown in cancer cells. Our data also support the hypothesis that glutamine is a likely alternative energy source during niacin deficiency. Here, we also identified the expression of all seven NAD-dependent deacetylase (SIRT) family members in skin cells. We showed that in response to photodamage, the expression of several SIRTs is altered in keratinocytes. Furthermore, we showed that SIRTs responses to photodamage differ between normal and immortalized keratinocytes, which may be indicative of alterations potentially important in skin carcinogenesis. In addition, we have shown that NAD-depleted HaCaT keratinocytes are more sensitive to photodamage. We observed that both poly(ADP-ribose) polymerases (PARPs) and SIRTs are inhibited by the unavailability of their substrate, NAD+, leading to unrepaired DNA damage upon photodamage and subsequent increase in cell death. Our data demonstrate that both SIRTs and PARPs are critical following DNA damage and identify which SIRTs are essential. Finally, we identified for the first time the expression of the nicotinic acid receptor in human skin keratinocytes, mainly in the differentiating keratinocytes of the stratum corneum in the epidermis. This study identifies new roles for niacin as a potential skin cancer prevention agent and demonstrates that niacin status is a critical resistance factor for UV damaged skin cells.