Elevated sympathetic nervous system (SNS) tone contributes to excess cardiac mortality associated with type 2 diabetes mellitus (T2DM). Chronic SNS stimulation has detrimental effects to the heart, in particular, with its cell signaling abilities. (R)-[11C]Rolipram small animal positron emission tomography (PET), an noninvasive nuclear imaging modality, was used to assess phosphodiesterase-4 (PDE4) alterations in a high fat diet (HFD), streptozotocin (STZ) induced model of hyperglycemia in rats. Prior to investigation in the animal model, characterization of (R)-[11C]rolipram small animal PET was completed. (R)-[11C]Rolipram binds specifically to PDE4 in the rat heart demonstrated by competitive blockade with (R)-rolipram with the PDE4 enzyme susceptible to saturation with increasing injected masses of unlabeled rolipram. (R)-[11C]Rolipram cardiac retention was elevated by acute norepinephrine stimulation via desipramine pharmacologic challenge. Quantitative (R)-[11C]rolipram PET was highly reproducible in the heart and presents an ideal avenue to investigate PDE4 alterations. (R)-[11C]rolipram small animal PET did not reveal changes in PDE4 expression and activity in STZ-treated hyperglycemic animals compared to STZ-treated euglycemic and control groups. In vitro measures of PDE4 enzyme expression and activity, with or without desipramine, were also not altered between treatment groups. Although (R)-[11C]rolipram small animal PET does not reveal PDE4 alterations in this animal model of diabetes, its utility to assess PDE4 alterations in other over active SNS pathologies, such as heart failure and obesity, remains.

Elevated sympathetic nervous system (SNS) tone contributes to excess cardiac mortality associated with type 2 diabetes mellitus (T2DM). Chronic SNS stimulation has detrimental effects to the heart, in particular, with its cell signaling abilities. (R)-[11C]Rolipram small animal positron emission tomography (PET), an noninvasive nuclear imaging modality, was used to assess phosphodiesterase-4 (PDE4) alterations in a high fat diet (HFD), streptozotocin (STZ) induced model of hyperglycemia in rats. Prior to investigation in the animal model, characterization of (R)-[11C]rolipram small animal PET was completed. (R)-[11C]Rolipram binds specifically to PDE4 in the rat heart demonstrated by competitive blockade with (R)-rolipram with the PDE4 enzyme susceptible to saturation with increasing injected masses of unlabeled rolipram. (R)-[11C]Rolipram cardiac retention was elevated by acute norepinephrine stimulation via desipramine pharmacologic challenge. Quantitative (R)-[11C]rolipram PET was highly reproducible in the heart and presents an ideal avenue to investigate PDE4 alterations. (R)-[11C]rolipram small animal PET did not reveal changes in PDE4 expression and activity in STZ-treated hyperglycemic animals compared to STZ-treated euglycemic and control groups. In vitro measures of PDE4 enzyme expression and activity, with or without desipramine, were also not altered between treatment groups. Although (R)-[11C]rolipram small animal PET does not reveal PDE4 alterations in this animal model of diabetes, its utility to assess PDE4 alterations in other over active SNS pathologies, such as heart failure and obesity, remains.

Elevated sympathetic nervous system (SNS) tone contributes to excess cardiac mortality associated with type 2 diabetes mellitus (T2DM). Chronic SNS stimulation has detrimental effects to the heart, in particular, with its cell signaling abilities. (R)-[11C]Rolipram small animal positron emission tomography (PET), an noninvasive nuclear imaging modality, was used to assess phosphodiesterase-4 (PDE4) alterations in a high fat diet (HFD), streptozotocin (STZ) induced model of hyperglycemia in rats. Prior to investigation in the animal model, characterization of (R)-[11C]rolipram small animal PET was completed. (R)-[11C]Rolipram binds specifically to PDE4 in the rat heart demonstrated by competitive blockade with (R)-rolipram with the PDE4 enzyme susceptible to saturation with increasing injected masses of unlabeled rolipram. (R)-[11C]Rolipram cardiac retention was elevated by acute norepinephrine stimulation via desipramine pharmacologic challenge. Quantitative (R)-[11C]rolipram PET was highly reproducible in the heart and presents an ideal avenue to investigate PDE4 alterations. (R)-[11C]rolipram small animal PET did not reveal changes in PDE4 expression and activity in STZ-treated hyperglycemic animals compared to STZ-treated euglycemic and control groups. In vitro measures of PDE4 enzyme expression and activity, with or without desipramine, were also not altered between treatment groups. Although (R)-[11C]rolipram small animal PET does not reveal PDE4 alterations in this animal model of diabetes, its utility to assess PDE4 alterations in other over active SNS pathologies, such as heart failure and obesity, remains.

Elevated sympathetic nervous system (SNS) tone contributes to excess cardiac mortality associated with type 2 diabetes mellitus (T2DM). Chronic SNS stimulation has detrimental effects to the heart, in particular, with its cell signaling abilities. (R)-[11C]Rolipram small animal positron emission tomography (PET), an noninvasive nuclear imaging modality, was used to assess phosphodiesterase-4 (PDE4) alterations in a high fat diet (HFD), streptozotocin (STZ) induced model of hyperglycemia in rats. Prior to investigation in the animal model, characterization of (R)-[11C]rolipram small animal PET was completed. (R)-[11C]Rolipram binds specifically to PDE4 in the rat heart demonstrated by competitive blockade with (R)-rolipram with the PDE4 enzyme susceptible to saturation with increasing injected masses of unlabeled rolipram. (R)-[11C]Rolipram cardiac retention was elevated by acute norepinephrine stimulation via desipramine pharmacologic challenge. Quantitative (R)-[11C]rolipram PET was highly reproducible in the heart and presents an ideal avenue to investigate PDE4 alterations. (R)-[11C]rolipram small animal PET did not reveal changes in PDE4 expression and activity in STZ-treated hyperglycemic animals compared to STZ-treated euglycemic and control groups. In vitro measures of PDE4 enzyme expression and activity, with or without desipramine, were also not altered between treatment groups. Although (R)-[11C]rolipram small animal PET does not reveal PDE4 alterations in this animal model of diabetes, its utility to assess PDE4 alterations in other over active SNS pathologies, such as heart failure and obesity, remains.

Dans la recherche de nouveaux traitements des maladies broncho-pulmonaires comme l'asthme et la broncho-pneumopathie chronique obstructive, les inhibiteurs de PDE4 sont des cibles intéressantes. Dans cette voie, notre laboratoire s'intéresse à la synthèse d'une nouvelle famille d'inhibiteurs à base pyridazinone. Pour cela, cette thèse couple la modélisation moléculaire (docking : développement méthodologique et application), la synthèse organique ainsi que des tests biologiques.Dans un premier temps, le développement du logiciel d'amarrage moléculaire AlgoGen a été poursuivi. AlgoGen (créé initialement à l'université de Lorraine en 2009) est un logiciel qui couple un algorithme génétique pour la recherche conformationnelle à une évaluation de l'énergie protéine-ligand à un niveau quantique semi-empirique alors que les autres logiciels existant effectuent ce calcul à un niveau classique en général. Le calcul d'une énergie à un niveau quantique est très coûteux en temps. C'est pourquoi, nous avons apporté de nombreuses modifications à ce logiciel afin d'accroître son efficacité dans la recherche conformationnelle. Ce logiciel a ensuite été utilisé sur un jeu de 22 dimères (typiques des reconnaissances moléculaires biologiques) et à huit systèmes protéine-ligand.Dans un deuxième temps, huit inhibiteurs ont été synthétisés et testés in vitro sur la cible PDE4. Pour compléter ce volet expérimental, une étude de structure-activité a été effectuée grâce au docking moléculaire (AlgoGen, Autodock, Glide) pour rationaliser les activités mesurées (IC50). Pour terminer, des pharmaco-modulations guidées par docking ont été réalisées afin de proposer de nouveaux inhibiteurs de plus grandes affinités avec la protéine PDE4D. / For the research of new treatment of bronchopulmonary diseases such as asthma and chronic obstructive pulmonary disease (COPD), the PDE4 inhibitors are an attractive target. Our laboratory is interested in a new PDE4 inhibitors family based on the pyridazinone pattern. For this purpose, this thesis couples molecular modeling (docking: methodological development and application), organic synthesis and biological tests.First, the development of the molecular docking software AlgoGen was continued. AlgoGen (initially created at university of Lorraine in 2009) is a program which couples a genetic algorithm for the conformational research and a protein-ligand energy evaluation at the quantum semi-empirical level while other software do this evaluation at a classical level. Quantum energy calculations are very time consuming. That is the reason why some modifications have been made to improve its efficiency for the conformational search. This software was then used for calculations on a set of 22 dimers (typical in biological molecular recognition) as well on 8 ligand-protein complexes.Secondly, eight inhibitors were synthesized and tested in vitro on the PDE4 target. To complete the experimental part, a structure-activity relationship study was carried out through a molecular docking to rationalize the measured activity (IC50). Finally, pharmaco-modulations guided by docking were made to propose new inhibitors with more affinity with the protein.

Vascular endothelial cells (VECs) play an essential role in regulating the passage of macromolecules and cells between the blood stream and underlying tissues. The second messenger 3’, 5’ cyclic adenosine monophosphate (cAMP) regulates numerous events in VECs, including permeability. Since human VECs express several distinct cAMP-hydrolyzing phosphodiesterases (PDEs), and these are the only enzymes that catalyze the inactivation of cAMP, we investigated if selective pharmacological inhibition of PDEs could impact VEC permeability. Interestingly, we found that PDE4 inhibitors decreased human aortic VEC (HAEC) permeability and PDE4 and PDE3 inhibitors decreased human microvascular VEC (HMVEC) permeability. Consistent with a role for both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in regulating VEC permeability, selective activators of these enzymes significantly decreased permeability. Since neither PDE4 nor PDE3 inhibitors significantly increased cAMP in these cells, our data are consistent with the idea that PDE inhibition causes small localized increases in “pools” of cAMP that regulate permeability. In order to test if PDE4 enzymes could act locally on pools of cAMP that regulated permeability, we selectively isolated the adherens junctional protein VE-cadherin from confluent monolayers of HAECs or HMVECs, and immunoblotted these isolates for cAMPeffectors and PDEs. Briefly, we found that each PKA-II, EPAC1, and a PDE4D variant, but not PDE3 enzymes, each could be isolated in VE-cadherin-based complexes from these cells. These novel findings identify PKA-II, EPAC1, and PDE4D as members of VE-cadherin-based signaling complexes in human VECs and are consistent with the idea that localized cAMP-signaling regulates permeability in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-14 15:52:20.216

Following the discovery of the DISC1 gene in 2000, subsequent research has led to DISC1 becoming one of the most promising candidate genes for psychiatric disorders. Acting as a scaffold protein, DISC1 has a large number of interacting proteins and is involved in a series of intracellular signalling pathways. Amongst these binding proteins are two enzymes, PDE4 and GSK3β, that were originally implicated in psychiatric disease by virtue of their inhibition by psychoactive drugs. PDE4 enzymes are inhibited by rolipram, which possesses anti-depressant and anti-psychotic activity, while GSK3β is one of the major targets of lithium, a potent mood stabiliser. Both these enzymes are intricately involved in the PI3K/AKT, cAMP, and MAPK signalling pathways, all of which have a number of downstream outcomes with potential relevance to psychiatric disorders. The Millar and Porteous laboratory had established that DISC1 modulates PDE4 activity, but this predated awareness of GSK3 as another DISC1 interactor whose binding site overlapped with that of PDE4. Since cAMP is a key regulator of signalling pathways in the brain, I hypothesised that not only DISC1, but also GSK3β may be involved in the regulation of PDE4 activity to control local cAMP levels and gradients. To investigate this hypothesis, I characterised SHSY5Y cells as a model for measuring PDE4 activity, and performed a series of genetic and pharmacological manipulations on this system. Inhibition of GSK3β resulted in a decrease of basal PDE4 activity that was amplified by DISC1 overexpression. Wild type cells that were treated with forskolin exhibited a significant increase in PDE4 activity, which was suppressed by GSK3β inhibition and both overexpression and knockdown of DISC1. Further experiments confirmed that none of these changes were a result of differences in PDE4 mRNA or protein expression. Thus I have provided evidence that suggests tonic activation of PDE4 by GSK3β and evidence for modulation of PDE4 activity by DISC1. I provide evidence for the localisation of PDE4B & PDE4D with key psychiatric associated receptors in structures resembling developing dendritic spines; furthermore, agonism of NMDA receptors results in a significant increase in PDE4 activity in primary neurons. These results are a simple demonstration of an emerging principle in psychiatric research: that none of the signalling pathways implicated in psychiatric disease are acting in isolation. There are likely to be multiple points of integration between these pathways, with the demonstrated DISC1-GSK3β-PDE4 interaction forming one of these points. My results add an important new element to the understanding of how the DISC1 complex may regulate intracellular signalling in response to extracellular cues.

Les antidépresseurs ont un effet antiallodynique qui dépend de la stimulation des récepteurs β2-adrénergiques. Ceux-ci stimulent la production d’adénosine monophosphate cyclique (AMPc) régulé par les phosphodiestérases de type 4 (PDE4). Nous avons ici étudié l’effet d’inhibiteurs de PDE (iPDE) sur la douleur neuropathique, grâce à des approches de pharmacologie comportementale chez la souris complétées par de l’imagerie calcium et des approches moléculaires. Nos résultats montrent un effet antiallodynique des iPDE4 et des iPDE5. L’action des iPDE4 est liée à une diminution d’expression du TNFα dans le ganglion rachidien et au recrutement des récepteurs delta des opioïdes. Celle des iPDE5 nécessite à la fois les récepteurs mu et delta. Nous montrons aussi que l’action d’un iPDE4 dépend de la dose, l’activation de cellules gliales semblant corrélée à l’effet antiallodynique à faible dose, alors que celle des neurones à forte dose a un effet pronociceptif via les récepteurs TRPV1. / Antidepressants have an antiallodynic action that is dependent on β2-adrenoceptor stimulation. These receptors stimulate the cAMP production, which is regulated by type 4 phosphodiesterases (PDE4). Here, we studied that action of PDE inhibitors (iPDE) on neuropathic pain, using behavioral pharmacology approaches in mice, completed by calcium imaging and molecular approaches. Our results show the iPDE4s and iPDE5s have an antiallodynic action. The iPDE4s act through a decreased expression of TNFα in dorsal root ganglia and the recruitment of the delta opioid receptors. The action of iPDE5 requires both mu and delta opioid receptors. We also show that the action of an iPDE4 depends on the dose, the activation of glial cells at low dose being correlated with an antiallodynic action, while the recruitment of neurons at higher doses has a pronociceptive action via TRPV1 receptors.

[pt] As reações de acoplamento C-C e C-N são metodologias muito úteis na funcionalização de estruturas orgânicas, principalmente no que tange a inserção de grupos arila em plataformas específicas. Chalconas são cetonas alfa, beta-insaturadas bastante exploradas na literatura devido à facilidade de obtenção e à estrutura facilmente modulável, o que faz com que as propriedades desses compostos possam ser dirigidas para objetivos específicos (chalconas bioativas, chalconas luminescentes, etc). Sua estrutura versátil permite utilizá-la como plataforma para reações regiosseletivas na porção olefínica e também permite funcionalizações nas porções arílicas. Para tal fim, reações de acoplamento carbono-carbono e carbono-nitrogênio catalisadas por metais de transição podem ser ferramentas de grande utilidade, especialmente na síntese de compostos poliarilados. O presente trabalho aborda a utilização de reações de acoplamento catalisadas por metais de transição na síntese de novas beta-arilchalconas, alfa, beta-diarilchalconas e chalconas N-diaril-substituídas na porção aromática. O trabalho é dividido em duas grandes partes: na primeira parte são abordadas as arilações de Heck e Suzuki na porção alceno de chalconas, com a obtenção de compostos cuja estrutura está de acordo com o farmacóforo proposto para inibidores alostéricos da enzima fosfodiesterase-4 (PDE4). Na segunda parte, a Reação de Ullmann é utilizada como etapa chave na obtenção de chalconas N-diariladas na porção aromática, levando a uma biblioteca de novos compostos fluorescentes. Em relação à arilação da porção alquenílica das chalconas, reações de Heck foram realizadas utilizando-se diferentes haletos aromáticos, resultando na obtenção de beta-arilchalconas com rendimentos variando de 20 a 85 porcento e relações E:Z de aproximadamente 75:25. A partir das misturas estereoisoméricas, obtevese isômeros majoritariamente E (E:Z maior 96:4) através de simples precipitações em solventes apropriados. Reações competitivas foram avaliadas por CG para a determinação da influência dos diferentes substituintes nos anéis aromáticos na velocidade da reação. alfa, beta-diarilchalconas foram obtidas a partir de intermediários beta-arilados, via metodologia sequencial de bromação seguida de acoplamento Suzuki, com rendimentos entre 60 e 99 porcento. Na segunda parte da dissertação é descrita a síntese de p-N-diarilaminochalconas, utilizando-se como etapa chave um acoplamento C-N catalisado por cobre. Foram obtidas chalconas com características D-n-D e D-n-A, além de beta-arilchalconas com características D-nD. Os compostos que apresentaram fluorescência mais intensa foram aqueles com característica D-n-D. A beta-arilação diminuiu consideravelmente a fotoluminescência dos compostos. A fluorescência dos compostos foi fortemente influenciada pelo solvente, sugerindo transferência de carga intramolecular no estado excitado. Com o objetivo de relacionar a estrutura dos compostos com suas características de emissão, foram realizados cálculos de DFT. / [en] C-C and C-N Coupling Reactions are useful methodologies for functionalization of organic structures, especially regarding the insertion of aryl groups in specific platforms. Chalcones are alpha, beta-unsaturated ketones widely explored in literature as a result of features such as easiness of preparation and highly modulating structure which allows the properties of these compounds to be directed for specific purposes (bioactive chalcones, luminescent chalcones, etc.) Its versatile structure allows it to be used as a platform for regioselective reactions in the olefin moiety and the structures also allows functionalization in the aryl fragment. For this purpose, the carbon-carbon and carbon-nitrogen reactions catalyzed by transition metals can be very useful tools, especially in the synthesis of polyarylated chalcones.The present work describes the use of metal-catalyzed coupling reactions in the synthesis of new beta-arilchalcones, alpha, beta-diarylchalcones and N-diaryl-substituted chalcones on the aromatic moiety. The manuscript is divided into two major parts: the first part deals with Heck and Suzuki Reactions in the alkene portion of chalcones, leading to compounds whose structures are in agreement with the proposed pharmacophore for allosteric inhibitors of the enzyme Phosphodiesterase 4 (PDE4). In the second part, the Ullmann Reaction is used as a key step in the synthesis of p-N-diarylated-chalcones, leading to a library of new fluorescent compounds.Regarding the arylation of the alkenyl moiety of chalcones, Heck reactions were carried out using different aromatic halides resulting in beta arylchalcones, in yields ranging from 20 to 85 porcento and E:Z ratios of approximately 75:25. From the stereoisomeric mixtures, the E-isomers could be isolated by simple precipitations in appropriate solvents. Competitive reactions were evaluated by GC to determine the influence of the different substituents on the aromatic rings over the reaction rate. alpha, beta-diaryl-chalcones were obtained from beta-aryl intermediates via sequential bromination/Suzuki coupling in yields of approximately 50 porcento. In the second part of this work the synthesis of diarylaminochalcones is described using a copper-catalyzed C-N coupling as the key step. Chalcones with D-n-D and D-n-A characteristics were obtained, as well as beta-arylchalcones with D-n-D design. The compounds that showed the most intense fluorescence were those with D-n-D characteristics. beta-arylation considerably decreases these compounds photoluminescence. Solvent effects were observed suggesting intramolecular charge transfer in the excited state. To relate the structure of the compounds with their emission characteristics, DFT experiments were performed.

The boronic acid functional group is known to bind compounds with the diol group tightly and reversibly in aqueous environment and has been used as a recognition moiety for the design of carbohydrate sensors. The first chapter of the dissertation studies the synthesis and substitution effect on the affinity and selectivity of a known boronic acid-based glucose sensor. In such a sensor design effort, the availability of a signaling event, whether it is fluorescence or UV, is crucial. The second chapter studies the detailed mechanism on how a well-known fluorescent boronic acid compound changes fluorescent properties upon binding. A new mechanism has been established which corrected a decade old mistake. In the third chapter, a series of boronic acid-based sensors were designed and synthesized for sialic acid, which is part of tetrasaccharide found on many cell surface carbohydrates. Such sialic acid sensors could be very useful for the development of new type of anti-influenza therapy. The fourth is on the design and synthesis novel and selective inhibitors for phosphodiesterase 4 (PDE4), which are potential anti-asthma agents.