Positron emission tomography (PET) tracer 3-fluoro-3-deoxythymidine (FLT) is used for imaging tumor proliferation. Prior to this work, human equilibrative nucleoside transporter 1 (hENT1) was the only known human nucleoside transporter (hNT) capable of FLT transport. The aim of this research was to determine if other hNTs, including hENT2, human concentrative nucleoside transporter 1 (hCNT1), hCNT2 and hCNT3, were capable/important of/for FLT transport in mammalian cells. Transport assays performed in Xenopus laevis oocytes producing recombinant hNTs demonstrated that hENT1/2 and hCNT1/3 were capable of FLT transport. FLT uptake assays with or without hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) in various cultured cancer cell lines demonstrated that hENT1 was responsible for the majority of mediated FLT uptake in all tested cell lines, suggesting that hENT1 was important for FLT uptake. The in vivo role of hENT1 in FLT uptake was determined by performing [18F]FLT PET on wild-type and ENT1 knockout mice. One hour after [18F]FLT injection, ENT1 knockout mice displayed significantly reduced [18F]FLT accumulation in the blood, heart, brain, kidney, liver, and lungs compared to wild-type mice. Interestingly, ENT1 knockout mice displayed increased [18F]FLT accumulation in the bone marrow and spleen which both have high CNT expression, suggesting that loss of ENT1 significantly alters FLT biodistribution in mice. hENT1 is a predictive marker of gemcitabine response in pancreatic cancers. Since FLT uptake and gemcitabine toxicity are dependent on hENT1, FLT uptake may predict gemcitabine response in pancreatic cancers. To test this hypothesis, six different pancreatic cancer cell lines were analyzed for FLT uptake and gemcitabine toxicity. hENT1/2 inhibition in cells decreased FLT uptake and gemcitabine sensitivity. In five of six cell lines, a positive correlation was observed between FLT uptake and gemcitabine toxicity, suggesting that FLT PET may be clinically useful for predicting gemcitabine response in pancreatic cancers. The results from this research suggest that hNTs, especially hENT1, are important for FLT uptake in mammalian cells and that FLT uptake can predict gemcitabine response in most cultured pancreatic cancer cells. The results warrant FLT PET clinical trials in pancreatic cancer patients to determine the potential of FLT PET in predicting gemcitabine response.

Hypoxic regions found in most solid tumors often contain cells which are resistant to various cancer therapies. However, hypoxia also forces cells to rely solely on the catabolism of glucose through glycolysis for ATP production and survival, thereby creating a therapeutic window that can be exploited by glycolytic inhibitors, such as 2-deoxy-D-glucose (2-DG). Previous studies in our lab demonstrated that activation of Hypoxia Inducible Factor (HIF-1) in hypoxic tumor cells confers resistance to glycolytic inhibition by 2-DG. In surveying a number of tumor types for differences in intrinsic levels of HIF-1 alpha under hypoxia, we found that pathways upstream of HIF -- i.e. AKT and mammalian target of rapamycin (mTOR) -- have significantly reduced activity in 2 human non-small lung cancer cell lines (NSCLC) as compared to 4 small cell lung cancer cell (SCLC) lines. This reduced activity of AKT and mTOR correlated with increased sensitivity to 2-DG under hypoxia. Since HIF-1 alpha translation is regulated by the mammalian target of rapamycin (mTOR), we examined the effects of blocking mTOR with an analog of rapamycin (CCI-779) in SCLC cells which express high levels of mTOR activity. Under hypoxia, treatment with CCI-779 resulted in HIF-1 alpha down-regulation. Furthermore, CCI-779 potentiated the cytotoxic effects of 2-DG in hypoxic SCLC cells. Conversely, CCI-779 did not increase 2-DG toxicity in NSCLC lines that do not express HIF, SCLC lines treated with siRNA against HIF-1 alpha, or HIF-deficient mutants. These latter results support the hypothesis that, although mTOR modulates numerous downstream pathways, mTOR inhibition by CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1 alpha. Overall, our findings show that CCI-779 hyper-sensitizes HIF-expressing hypoxic tumor cells to 2-DG. Additionally, our results suggest that the intrinsic expression of AKT, mTOR, and HIF in many tumor types may be important predictors of clinical responsiveness to 2-DG and could be used to guide future treatment decisions on whether to use 2-DG alone or in combination with an mTOR inhibitor.

No description available.

Orientador: Cleslei Fernando Zanelli / Banca: Nilson Ivo Tonin Zanchin / Banca: Paulo Sergio Rodrigues Coelho / Resumo: O fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e é essencial para a viabilidade celular. Este fator é a única proteína conhecida que sofre uma modificação pós-traducional única e necessária para a função de eIF5A, em que uma lisina específica é convertida em um resíduo de hipusina pela ação das enzimas desoxi-hipusina sintase (Dys1) e desoxi-hipusina hidroxilase (Lia1). Inicialmente, eIF5A foi relacionada à etapa do início da tradução, porém, dados recentes sugerem a sua atuação na etapa de elongação ao invés de início. No entanto, além do fato de a função específica de eIF5A na célula não ser conhecida, o papel da hipusinação para o funcionamento de eIF5A também não é conhecido. Diante disso, o objetivo deste trabalho é caracterizar mutantes condicionais para o gene da desoxi- hipusina sintase e, dessa forma, contribuir para o entendimento não só da função da hipusinação sobre eIF5A, mas também para o entendimento da função específica de eIF5A na célula. Para isso, foram iniciadas análises de caracterização fenotípica com os alelos dys1Δ1-28 e dys1W75R/T118A/A147T (dys1-1). Inicialmente, foi realizada a subclonagem do alelo dys1Δ1-28 , uma vez que, por ter sido identificado em um rastreamento de duplo-híbrido, este alelo estava em fusão com a região codificadora do domínio de ativação de Gal4. Porém, após realização da subclonagem, ou seja, quando na ausência do domínio de ativação, este alelo não apresentou o fenótipo condicional de crescimento inicialmente observado. Portanto, o mutante se tornou impróprio para a realização dos ensaios subsequentes e foi descartado. Em seguida, foram iniciadas as análises com o alelo dys1-1, nas quais foi observada diminuição nos níveis totais de Dys1 mutada, e consequentemente, diminuição nos níveis de hipusinação. Devido a isso ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and is essential for cell viability. This factor is the only known protein that undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into hypusine by the action of the enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1). Initially, eIF5A was related to the initiation step of translation, however, recent data suggest a role in the elongation step of translation. However, besides the fact that the specific function of eIF5A in the cell is still obscure, the role of hypusination in eIF5A function is unknown. Thus, the goal of this project is to characterize conditional mutants of the deoxyhypusine synthase gene and thereby contribute to the understanding not only the function of hypusination in eIF5A, but also of the specific role of eIF5A in the cell. We started a phenotypic characterization of two different alleles: dys1Δ1-28 and dys1W75R/T118A/A147T (dys1-1). Initially, we performed a subcloning of the allele dys1Δ1-28 , once the allele was fused with the coding region of GAL4 activation domain, due to the fact that this allele is devived of a two-hybrid screening. However, after performing the subcloning, that is, in the absence of the activation domain, this allele showed no conditional growth phenotype as originally observed. Therefore, this mutant has become improper to carry out the subsequent analysis and was discarted. Then, the analyses with dys1-1 allele were initiated, in which it was observed a decrease in total levels of Dys1 and, consequently, a decrease in the hypusination levels. Because of that, this allele shows a decrease in cell growth rate and growth arrests after 24 hours in medium lacking the osmotic regulator. However, this growth arrest is not followed by cell lysis. Furthermore, the mutant ... (Complete abstract click electronic access below) / Mestre

No description available.

Background. Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy. Methods. Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-d-glucose [2-DG], 0.25-2 g/kg, n = 6-8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. Results. Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG-treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1β as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium. Conclusions. Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

Molecular drug design begins with the identication of a problem to solve. This work identies the growing resistance among human pathogens to current treatments. Once the problem is identied and understood, solutions must be proposed. This one is straight forward, we need new antimicrobial drugs. More specically, we need to identify novel targets to inhibit. A large portion of antibiotics focus on disruption of macromolecular production while only a few target metabolic systems. Finally, you need to propose solutions based on the information gathered. In order to avoid existing resistance, it is important to avoid the macromolecular route and focus on metabolic enzymes. Preferably, the pathway would have little overlap or similarity with pathways found in the treatment organism. With this in mind, the non-mevalonate (NMA) pathway poses as a very good target for drug design. Many pathogens have been found to be strictly dependent on this pathway while it is absent in humans. Additionally, fosmidomycin has already been shown to inhibit this pathway. Initially thought to just inhibit the 1-deoxy-D-xylulose 5- phosphate (DXP) reductoisomerase (DXR), it has been shown to inhibit several enzymes along the path to a lesser extent. Ideally, this could be repeated or improve upon for future drug design. With this in mind, the initial stages of the rst two enzymes of the NMA pathway were examined utilizing quantum mechanical/molecular mechanical (QM/MM) techniques. The rst enzyme was DXP synthase (DXS), which catalyzes a transketolase-like condensation of pyruvate and glyceraldehyde-3-phosphate to produce DXP. DXS and other transketolases are dependent on the thiamine diphosphate (TDP) cofactor, which must be deprotonated of the imidazolium C2 atom producing a highly reactive ylide. A tautomerization occurs prior to this deprotonation to prime the pyrimidinium ring N4 atom to perform the C2 abstraction. The question at hand was the identity of a general base to perform the N4 abstraction. The results favored a water-mediate mechanism with a higher than usual Ez of 22.7 kcal/mol. An observation pertaining the tautomerization pertained to the aromaticity of the pyrimidine ring. Upon further investigation, aromaticity was found to play a signicant role in the ΔE observed. Aromaticity might contribute 14.2 kcal/mol to the barrier height. This high energy would drive the reaction forward producing the ylide. Investigation of the DXR enzyme followed this work. Initially, the work was going to focus on the 2 mechanisms proposed for activity, alpha-ketol rearrangement and retroaldol/ aldol mechanism. Subsequent publications involving secondary kinetic isotope effects (KIEs) add to the pile of evidence supporting the retro-aldol/aldol mechanism. So the project was retooled to investigate the energetic dierences between two metal binding modes. The results of this work support a metal coordination across the C3-C4 bond, which eventually extends coordination to include the C2 oxygen. This conformation was help explain the tight binding eecting observation of the putative intermediates (transition states) and aldehyde intermediate. Additionally, as the C2-C3 mode consistently transfers a proton to the phosphate group of DXP or produces an elongated C-O bond, the C2-C3 mode would not be favorable. Further investigations of these enzymes (e.g. completing the step begin, continuing through the reaction) could provide further illumination into the mechanism of action and possibly reveal new avenues of drug design. Examining the enzymes downstream in the NMA pathway might provide details of interest. Of particular interest is the radical reaction proposed for HDR/IspH. The nal step of the pathway produces IDP and DMADP in a 4:1 proportion, which corresponds to the general system requirements for production of the long chain, branched isoprenoids. It would be interesting to compute the mechanism to see if energetics could provide further insights. Additionally, normal mode analysis coupled with vibrational subsystem analysis could identify allosteric sites for feedback sensitivity.

La voie non-mévalonate est fortement présente chez les plantes et les bactéries mais est absente chez les mammifères. C'est pourquoi inhiber la synthèse des isoprénoïdes et identifier un inhibiteur de cette voie enzymatique contribuera grandement à la recherche de nouveaux antibiotiques, antifongiques et herbicides. Les propriétés uniques de la 1-deoxy-D-xylulose 5-phosphate reductoisomérase (DXR), l'enzyme centrale de cette voie enzymatique, en font une cible très intéressante pour la synthèse de nouveaux composés. La Fosmidomycine agit comme un inhibiteur de la DXR et reste aujourd'hui, avec son homologue acétylé FR90098, la référence en termes d'inhibiteur même si de nombreux efforts ont été faits pour la synthèse d'analogues depuis plusieurs années comme expliqué dans le premier chapitre avec la mise en relation de la structure des composés et leur activité. L'analyse de la diffraction des rayons X de la DXR avec la Fosmidomycine où le substrat naturel montre que la fonction phosphonate ou phosphate interagit avec une poche polaire hautement spécifique dans le site actif de l'enzyme permettant peu de modifications. Par comparaison, la fonction acide hydroxamique qui chélate le cation de l'enzyme offre la possibilité de modifications par l'introduction d'autres fonctions complexantes. Dans ce contexte, de nombreuses modifications comme l'introduction de fonctions carbamoylphosphinate, amidoxime, N-hydroxyurée et dérivées d'uraciles comme unités complexantes ont été synthétisées pour trouver des nouvelles familles d'inhibiteurs de la DXR. Toutes ces fonctions possèdent des propriétés de chélation intéressantes. En effet, elles ont déjà conduit à de puissants inhibiteurs de différentes métalloenzymes. / The non-mevalonate pathway is highly present in higher plants, protozoa and bacteria but as no equivalent in mammals. That is why shut down isoprenoid biosynthesis and identify a non-mevalonate pathway inhibitor would greatly contribute to the search for safer antibiotics, antimalarials and for our concern herbicides. The unique properties of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the central enzyme of this pathway, make it a remarkable and attractive target for drug design. Fosmidomycin acts as an inhibitor of DXR and still remains, along with its N-acetyl homologue FR90098, one of the most potent inhibitor ever known even if extensive work on the development of Fosmidomycin analogue derivatives have been developed since the last decade as demonstrated in the first chapter with the development of a structure activity relationship of all the potential inhibitors of this enzyme already reported in the literature. The X-ray diffraction analysis of the co-crystals of DXR and Fosmidomycin or substrate shows that the phosphonic/phosphate group interacts with a highly specific polar pocket in the enzyme site, allowing only few structural modifications. By contrast, the cation chelating subunit represented by the hydroxamic acid function offers fine tuning possibilities for the complexation abilities as well as potential secondary interactions with the NADPH cofactor or directly with the enzyme. In this context, several modifications such as the introduction of carbamoylphosphinate, amidoxime, N-hydroxyurea and uracil complexing subunits have been made in order to find new families of DXR inhibitors. All of these functions show promising chelation capabilities as they already led to potent inhibitors of different metalloenzymes.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.