cGMP-dependent protein kinases (PKG) are key players in intracellular second messenger signaling within many cellular systems throughout the body. Most notably PKG is known for its role in smooth muscle relaxation (Pfeiffer et.al, 1999). The Iα PKG isozyme has been identified as the primary effector of the nitric oxide pathway (and serves to be a novel drug target). To date the overall knowledge of structure and function of PKG is lacking in terms of the mechanisms of activation and the structural orientations that coordinate them. Recently, our laboratory has solved the crystal structure of the regulatory domain of PKG Iα, which revealed a previously unknown α-helical domain dubbed the Switch Helix (SW) (Osborne et.al, 2011). The SW domain was found to be a site of interprotomer communication via hydrophobic interactions between its C-terminus and hydrophobic residues, named the nest located on the opposing protomer. Synthetic peptides derived from the SW domain, named S-tides, dosedependently activate PKG Iα (Moon et.al, 2015). In addition, the amino acid residues of the nest are in proximity to the cGMP binding site B. It was hypothesized that the binding site for S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. The hypothesis of this thesis is the binding site for the S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. To test this hypothesis two aims were constructed: Aim 1: To develop an S1.5 analog that utilizes both the nest and the B-site to increase S-tide activity, Aim 2: To explore the intricacies of these modes of activation and how they interact with each other to obtain a better understanding of the interplay between these two sites. First, based on the most potent S-tide S1.5 (YEDAEAKAKYEAEAAFFANLKLSD, Ka=6 μM), two analogs were synthesized. The peptide S2.5 which lacked the amino acids LSD at the C-terminus showed a three-fold lower activation constant (Ka= 15 μM), although the molecule retained its helicity as demonstrated by circular dichroism. The second analog, S3.5 contained unnatural amino acid components from a molecular modeling approach in an effort to further increase the affinity by interacting with the adjacent cGMP binding site B. However, S3.5 showed further reduction in activity with an activation constant of 70 μM. These findings led us to conclude that the failure of the SAR approach indicates a different mode of S-tide activation as had been previously thought. Next, we investigated the role of the cGMP binding site B in the mechanism of S-tide mediated PKG Iα activation. Co-activation assays with cGMP and S1.5 demonstrated that cGMP activation is not altered in the presence of S1.5. Furthermore, S1.5 mediated activation is negatively affected in the presence of cGMP. These results suggest that the B-site of cGMP does not positively enforce the S1.5 activation kinetics. Next, we employed the PKG Iα mutant E292A, which cannot bind cGMP to the B-site (Moon et.al., 2018). Interestingly, this mutant retains the activation kinetics of PKG Iα WT when activated via S1.5 and cGMP. Thus, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with cGMP. Likewise, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with S1.5. To further support these findings, the PKG Iα mutant C42A, which showed crippled cGMP activation kinetics could be activated with S1.5 with a potency similar to wild type. Taken together, the results in this thesis demonstrate that in contrast to the initial hypothesis the binding sites for S-tides and cGMP, although in proximity, show no experimental support of a positive interaction. These findings are significant as they reveal that S1.5 mediated activation of PKG is truly independent of cGMP, thereby providing a molecular platform for the therapeutic development of these unique peptides.

Actin-based directional motility is important for embryonic development, wound healing, immune responses, and development of tissues. Actin and myosin are essential players in this process that can be subdivided into protrusion, adhesion, and traction. Protrusion is the forward movement of the membrane at the leading edge of the cell. Adhesion is required to enable movement along a substrate, and traction finally leads to the forward movement of the entire cell body, including its organelles. While actin polymerization is the main driving force in cell protrusions, myosin motors lead to the contraction of the cell body. The goal of this work was to study the regulatory mechanisms of the motile machinery by selecting a representative key player for each stage of the signaling process: the regulation of Arp2/3 activity by WASP (actin system), the role of cGMP in myosin II assembly (myosin system), and the influence of phosphoinositide signaling (upstream receptor pathway). The model organism chosen for this work was the social ameba Dictyostelium discoideum, due to the well-established knowledge of its cytoskeletal machinery, the easy handling, and the high motility of its vegetative and starvation developed cells. First, I focused on the dynamics of the actin cytoskeleton by modulating the activity of one of its key players, the Arp2/3 complex. This was achieved using the carbazole derivative Wiskostatin, an inhibitor of the Arp2/3 activator WASP. Cells treated with Wiskostatin adopted a round shape, with no of few pseudopodia. With the help of a microfluidic cell squeezer device, I could show that Wiskostatin treated cells display a reduced mechanical stability, comparable to cells treated with the actin disrupting agent Latrunculin A. Furthermore, the WASP inhibited cells adhere stronger to a surface and show a reduced motility and chemotactic performance. However, the overall F-actin content in the cells was not changed. Confocal microscopy and TIRF microscopy imaging showed that the cells maintained an intact actin cortex. Localized dynamic patches of increased actin polymerization were observed that, however, did not lead to membrane deformation. This indicated that the mechanisms of actin-driven force generation were impaired in Wiskostatin treated cells. It is concluded that in these cells, an altered architecture of the cortical network leads to a reduced overall stiffness of the cell, which is insufficient to support the force generation required for membrane deformation and pseudopod formation. Second, the role of cGMP in myosin II dynamics was investigated. Cyclic GMP is known to regulate the association of myosin II with the cytoskeleton. In Dictyostelium, intracellular cGMP levels increase when cells are exposed to chemoattractants, but also in response to osmotic stress. To study the influence of cyclic GMP on actin and myosin II dynamics, I used the laser-induced photoactivation of a DMACM-caged-Br-cGMP to locally release cGMP inside the cell. My results show that cGMP directly activates the myosin II machinery, but is also able to induce an actin response independently of cAMP receptor activation and signaling. The actin response was observed in both vegetative and developed cells. Possible explanations include cGMP-induced actin polymerization through VASP (vasodilator-stimulated phosphoprotein) or through binding of cGMP to cyclic nucleotide-dependent kinases. Finally, I investigated the role of phosphoinositide signaling using the Polyphosphoinositide-Binding Peptide (PBP10) that binds preferentially to PIP2. Phosphoinositides can recruit actin-binding proteins to defined subcellular sites and alter their activity. Neutrophils, as well as developed Dictyostelium cells produce PIP3 in the plasma membrane at their leading edge in response to an external chemotactic gradient. Although not essential for chemotaxis, phosphoinositides are proposed to act as an internal compass in the cell. When treated with the peptide PBP10, cells became round, with fewer or no pseudopods. PH-CRAC translocation to the membrane still occurs, even at low cAMP stimuli, but cell motility (random and directional) was reduced. My data revealed that the decrease in the pool of available PIP2 in the cell is sufficient to impair cell motility, but enough PIP2 remains so that PIP3 is formed in response to chemoattractant stimuli. My data thus highlights how sensitive cell motility and morphology are to changes in the phosphoinositide signaling. In summary, I have analyzed representative regulatory mechanisms that govern key parts of the motile machinery and characterized their impact on cellular properties including mechanical stability, adhesion and chemotaxis. / Das Ziel der Arbeit war es, die regulatorischen Mechanismen der Zellmotilität zu untersuchen. Dazu habe ich für jedes Stadium dieses Prozesses einen repräsentativen regulatorischen Schritt ausgewählt und genauer untersucht: Die Regelung des Arp2/3 Komplexes durch WASP (Aktinsystem), die Rolle von cGMP in der Myosin II-Regulation (Myosinsystem) und der Einfluss von Phosphoinositiden im intrazellulären Signalprozess (Rezeptor-Signalweg). Die soziale Amöbe Dictyostelium discoideum wurde als Modellorganismus für diese Arbeiten gewählt. Gründe für diese Wahl waren die bereits vorliegenden detaillierten Kenntnisse über das Zytoskelett dieser Zellen, ihre einfache Handhabbarkeit im Labor, und die hohe Motilität der Zellen im vegetativen und entwickelten Zustand. Als Erstes analysierte ich die Dynamik des Aktin-Zytoskeletts durch Modulation der Aktivität des Arp2/3-Komplexes. Dafür benutzte ich das Carbazol-Derivat Wiskostatin, ein Inhibitor des Arp2/3-Aktivators WASP. Zellen, die mit Wiskostatin behandelt wurden, zeigten eine runde Form mit wenigen oder keinen Pseudopodien. Mit Hilfe des mikrofluidischen cell squeezer device konnte ich zeigen, dass Wiskostatin-behandelte Zellen eine geringere mechanische Stabilität aufweisen, vergleichbar mit Zellen unter dem Einfluss des Aktin-depolymerisierenden Wirkstoffes Latrunculin A. Darüber hinaus zeigen Wiskostatin behandelten Zellen eine erhöhte Substratadhäsion und eine verringerte Motilität und chemotaktische Effizienz. Der F-Aktingehalt der Zelle insgesamt blieb jedoch unverändert. Konfokal- und TIRF-mikroskopische Aufnahmen zeigten, dass die Zellen einen intakten Aktinkortex aufweisen. Es konnten lokalisierte dynamische Regionen erhöhter Aktinpolymerisation beobachtet werden, die jedoch nicht zur Ausbildung von Membrandeformationen führten. Daraus kann man rückschließen, dass die Mechanismen der Krafterzeugung im Aktin-Zytoskelett in WASP-inhibierten Zellen beeinträchtigt sind. Vermutlich liegt in diesen Zellen eine veränderte Mikroarchitektur des kortikalen Netzwerks vor, die zu einer verminderten Steifigkeit der Zelle führt, so dass die zur Bildung von Pseudopodien erforderlichen Kräfte nicht entfaltet werden können. Als Zweites wurde die Rolle von cGMP in der Myosin II-Dynamik untersucht. Es ist bekannt, dass cGMP die Assoziation von Myosin II mit dem Zytoskelett reguliert. In Dictyostelium steigt die intrazelluläre Konzentration von cGMP in Gegenwart von chemoattraktiven Lockstoffen sowie in Antwort auf osmotischen Stress. Um den Einfluss von cGMP auf die Aktin und Myosin II -Dynamik zu untersuchen, benutzte ich laserinduzierte Photoaktivierung von DMACM-caged-Br-cGMP, um cGMP lokal innerhalb der Zelle freizusetzen. Meine Ergebnisse zeigten, dass intrazelluläres cGMP direkt zur Aktivierung von Myosin II führt, jedoch auch Aktinantworten unabhängig vom cAMP-Rezeptorsignalweg induzieren kann. Die Aktinreaktion wurde sowohl in vegetativen als auch in entwickelten Zellen beobachtet. Eine mögliche Erklärung könnte die cGMP-induzierte Aktinpolymerisation über VASP (vasodilator-stimulated phosphoprotein) sein oder über die Bindung von cGMP an Nukleotid-abhängige Proteinkinasen. Als dritten Punkt meiner Arbeit untersuchte ich die Rolle der Phosphoinositide mit Hilfe des Phosphoinositide-bindenden Proteins PBP10, das bevorzugt an PIP2 bindet. Phosphoinositiden können Aktin-bindende Proteine zu definierten subzellulären Orten rekrutieren und ihre Aktivität verändern. Sowohl Neutrophile als auch entwickelte Dictyostelium Zellen produzieren PIP3 in der Plasmamembran an ihrer leading edge in Antwort auf externe Gradienten chemischer Lockstoffe. Obwohl Zellen auch ohne PIP3 chemotaktisches Verhalten zeigen, werden Phosphoinositide im Allgemeinen mit dem inneren chemotaktischen Kompass der Zelle in Verbindung gebracht. Mit dem Peptid PBP10 behandelte Zellen nahmen eine runde Form an, mit wenigen oder keinen Pseudopodien. PH-CRAC -Translokation zur Membran konnte in PBP10-behandelten Zellen selbst bei geringen cAMP-Stimuli weiterhin beobachtet werden. Ungerichtete wie auch gerichtete Zellmotiliät waren jedoch beeinträchtigt. Meine Daten zeigen, dass die Abnahme des PIP2-Pools in der Zelle durch PBP10 ausreicht, um die Zellmotilität zu beeinträchtigen, dass jedoch genug PIP2 erhalten bleibt um in Folge einer Rezeptorstimulation PIP3 zu produzieren. Die Ergebnisse demonstrieren daher, wie empfindlich Zellmotilität und -morphologie gegenüber Modifikationen im Phosphoinositid-Signalweg sind. Zusammenfassend habe ich mehrere repräsentative Beispiele für regulatorische Mechanismen der Zellmotilität untersucht und deren Auswirkung auf Eigenschaften der Zelle wie mechanische Stabilität, Zelladhäsion und Chemotaxis charakterisiert.

The neurobiology of anxiety-related disorders is associated with impaired neuroplasticity. The glutamate/NO/cGMP pathway has been proposed to play a key role in neuroplasticity and neurodevelopment. It was demonstrated in recent reports that chronic co-administration of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the antimuscarinic agent atropine exerts antidepressive-like activity in rats, and that this effect is related to PDE5 inhibition, with consequent elevation of cGMP levels and enhanced protein kinase G stimulation. The current study investigated possible anxiolytic effects of the chronic co-administration of sildenafil and atropine in stress-sensitive Flinders Sensitive Line (FSL) rats. FSL rats received vehicle control, fluoxetine (15 mg/kg), atropine (1 mg/kg), sildenafil (10 mg/kg) or sildenafil plus atropine via intraperitoneal administration, either acutely 30 minutes prior to testing (acutely) or daily for 14 days (chronically). FRL control rats received only vehicle. Thereafter anxiety-like behaviour was evaluated in the social interaction test (SIT - acute) and elevated plus maze (EPM - acute and chronic). The current study also compared to different ways to score the EPM, namely the percentage time spend in the open arms of the EPM and both the number of full and half body open arm entries, and also implemented defecation on the EPM as a measure of anxiety. Vehicle-treated FSL rats exhibited more anxiety-like behaviour than FRL rats in both the SIT and EPM following acute treatment, and in the EPM following chronic treatment. Acute treatment with fluoxetine exerted anxiogenic activity in the SIT and EPM, but anxiolytic activity following chronic administration, as observed in the EPM. In acute treatments neither sildenafil nor sildenafil plus atropine yielded any significant effects on anxiety-like behaviour. However, following chronic treatment, sildenafil exerted anxiolytic activity in the EPM by increasing the time spend in the open arms (45.72% ± 9.94% vs. 20.80% ± 9.94%, P<0.001). Atropine exerted a small anxiolytic response (30.71% ± 8.40% vs. 20.80 ± 9.94%), whereas atropine co-administration was additive to sildenafil alone and yielded an enhanced anxiolytic effect in the elevated plus maze (59.56% ± 4.95% vs. 20.80% ± 9.94%, P<0.001), relative to vehicle control. The percentage time spend in the open arms was scored in the EPM, the results suggested that the chronic treatment with sildenafil plus atropine exert an anxiolytic-like effect in FSL rats and the number of fecal droppings did not increase which is also an indication of an anxiolytic-like effects of the treatment. The current study demonstrated that the chronic treatment with sildenafil, alone or in combination with atropine, exhibit an anxiolytic-like action in stress-sensitive rats. In addition, the data support the clinical potential of using PDE5 inhibitors as antidepressant and anxiolytic strategy and warrant further investigation. Furthermore the study supports the previously proposed key role of the glutamate/NO/cGMP pathway in the neurobiology of anxiety-like disorders, and as an important target for drug development. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011

Appetite control and satiety mechanisms help animals maintain energy homeostasis; however, these mechanisms can be misregulated, leading to overweight and obesity. Caenorhabditis elegans is an excellent model system to study appetite and satiety because of its conserved behavioral aspects of satiety and conserved molecular mechanisms. ASI senses nutrition and its activity is required for the behavioral state of satiety quiescence. The purpose of this thesis project was to elucidate the function of cGMP signaling in ASI by looking at behavioral effects from the pharmacological use of sildenafil (Viagra), a PDE inhibitor, and the effects on ASI activation from mutating guanylyl cyclase DAF-11. Sildenafil treatment increases satiety quiescence and decreases fat storage in a PDE-dependent manner. The daf-11 mutation decreased overall fluorescence intensity of ASI activation and the frequency at which ASI activated by about 50% compared to wild-type worms, suggesting that DAF-11 plays an important role in ASI to promote satiety.

Master of Science / Food Science Institute / Fadi Aramouni / The brewing industry has, historically, had little food safety regulation. In response to the September 11th attacks, new legislation culminating in the Food Safety Modernization Act of 2010 was established and reinforced food safety regulations for the brewing industry. Under this expanded regulation, breweries are required to comply with the modernized Current Good Manufacturing Practices (cGMPs). The regulatory climate for the brewing industry is very complicated. The brewing industry is regulated primarily under the jurisdiction of the Alcohol and Tobacco Tax and Trade Bureau and the Food and Drug Administration. Based upon the regulatory environment and business considerations, Boulevard Brewing Company has opted to develop a Hazard Analysis and Critical Control Point (HACCP) plan. Before a HACCP plan can be implemented a foundation of prerequisite programs, based on the FDA’s cGMPs, must be in place. Prerequisite programs establish the operational and environmental conditions required for a successful HACCP plan. Failure to comply with the cGMPs can lead to fines, re‐inspection fees, forced recalls, and possible criminal prosecution. Prerequisite programs were developed for the following areas: facilities, including sanitary design principles, utilities, traffic and product flow; production equipment, including preventive maintenance and calibration; receiving, warehousing, and shipping, including supplier control, chemical control, and raw material testing; pest control for insects, rodents and birds; cleaning and sanitation under a Master Sanitation Schedule; specifications, including ingredients, products, and packaging materials; personal hygiene for both employees and visitors; and lastly a system of traceability and recall. These prerequisite programs, based on cGMPs, are required to ensure regulatory compliance while minimizing regulatory and fiscal risks.

Natriuretic peptides are a group of hormones, including atrial-, brain-, and C-type- natriuretic peptides (ANP, BNP, CNP). BNP can bind to two NP receptors (NPRs) denoted NPR-A (activates guanylyl cyclase) and NPR-C (activates inhibitory G- proteins). This study investigated the electrophysiological effects of BNP on isolated mouse atrial myocytes. Current-clamp experiments show that BNP had no effect on action potential (AP) parameters in basal conditions; however, when pre-stimulated with the ?-adrenergic receptor agonist isoproterenol (ISO), BNP prolonged AP duration. Voltage-clamp experiments demonstrate that BNP increased L-type calcium current (ICa,L) in the presence of ISO without altering cardiac potassium currents. The BNP effect on ICa,L was blocked by A71915 (a selective NPR-A antagonist), maintained in myocytes lacking NPR-C receptors and blocked by the phosphodiesterase-3 (PDE-3) inhibitor milrinone. These data demonstrate that BNP prolongs AP duration and increases ICa,L in atrial myocytes by activating NPR-A, increasing intracellular cGMP, and inhibiting PDE- 3.

The neurobiology of anxiety-related disorders is associated with impaired neuroplasticity. The glutamate/NO/cGMP pathway has been proposed to play a key role in neuroplasticity and neurodevelopment. It was demonstrated in recent reports that chronic co-administration of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the antimuscarinic agent atropine exerts antidepressive-like activity in rats, and that this effect is related to PDE5 inhibition, with consequent elevation of cGMP levels and enhanced protein kinase G stimulation. The current study investigated possible anxiolytic effects of the chronic co-administration of sildenafil and atropine in stress-sensitive Flinders Sensitive Line (FSL) rats. FSL rats received vehicle control, fluoxetine (15 mg/kg), atropine (1 mg/kg), sildenafil (10 mg/kg) or sildenafil plus atropine via intraperitoneal administration, either acutely 30 minutes prior to testing (acutely) or daily for 14 days (chronically). FRL control rats received only vehicle. Thereafter anxiety-like behaviour was evaluated in the social interaction test (SIT - acute) and elevated plus maze (EPM - acute and chronic). The current study also compared to different ways to score the EPM, namely the percentage time spend in the open arms of the EPM and both the number of full and half body open arm entries, and also implemented defecation on the EPM as a measure of anxiety. Vehicle-treated FSL rats exhibited more anxiety-like behaviour than FRL rats in both the SIT and EPM following acute treatment, and in the EPM following chronic treatment. Acute treatment with fluoxetine exerted anxiogenic activity in the SIT and EPM, but anxiolytic activity following chronic administration, as observed in the EPM. In acute treatments neither sildenafil nor sildenafil plus atropine yielded any significant effects on anxiety-like behaviour. However, following chronic treatment, sildenafil exerted anxiolytic activity in the EPM by increasing the time spend in the open arms (45.72% ± 9.94% vs. 20.80% ± 9.94%, P<0.001). Atropine exerted a small anxiolytic response (30.71% ± 8.40% vs. 20.80 ± 9.94%), whereas atropine co-administration was additive to sildenafil alone and yielded an enhanced anxiolytic effect in the elevated plus maze (59.56% ± 4.95% vs. 20.80% ± 9.94%, P<0.001), relative to vehicle control. The percentage time spend in the open arms was scored in the EPM, the results suggested that the chronic treatment with sildenafil plus atropine exert an anxiolytic-like effect in FSL rats and the number of fecal droppings did not increase which is also an indication of an anxiolytic-like effects of the treatment. The current study demonstrated that the chronic treatment with sildenafil, alone or in combination with atropine, exhibit an anxiolytic-like action in stress-sensitive rats. In addition, the data support the clinical potential of using PDE5 inhibitors as antidepressant and anxiolytic strategy and warrant further investigation. Furthermore the study supports the previously proposed key role of the glutamate/NO/cGMP pathway in the neurobiology of anxiety-like disorders, and as an important target for drug development. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011

Herzinsuffizienz ist ein weltweites Gesundheitsproblem mit hoher Morbidität und Mortalität und immer noch schlechter Prognose. Ein charakteristisches Merkmal der molekularen und damit verbundenen strukturellen Veränderungen, die der terminalen Insuffizienz vorangehen ist die durch Desensitivierungsmechanismen vermittelte Abnahme des beta-adrenergen (β-AR) Signalmoleküls zyklisches Adenosinmonophosphat (cAMP) auf der einen Seite und der gleichzeitigen Zunahme des von natriuretischen Peptiden (NP) und Stickstoffmonoxid (NO) generierten zyklischen Guanosinmonophosphat (cGMP) auf der anderen Seite. Während hohe cAMP-Spiegel im Herzen als schädlich gelten, werden cGMP-abhängige Signalkaskaden vorwiegend als protektiv verstanden. Amplitude, Lokalisation und Halbwertszeit beider Signalmoleküle werden durch spezifische Enzyme, den Phosphodiesterasen (PDE) reguliert. Unter der PDE-Superfamilie wird die Isoform PDE2 als einzige von cGMP aktiviert, um dann verstärkt cAMP abzubauen und steht damit im Zentrum eines negativen Crosstalks dieser beiden Signalwege. PDE2 ist sowohl in der humanen als auch der experimentellen Herzinsuffizienz hochreguliert und scheint dort am β-AR Desensitivierungsprozess beteiligt zu sein. Im Rahmen dieser Arbeit wurde die pathophysiologische Rolle der PDE2 im Herzen näher charakterisiert. Es wird gezeigt, dass die PDE2 nicht nur in Kardiomyozyten, sondern auch in kardialen Fibroblasten exprimiert wird. In Fibroblasten inhibieren cAMP/cGMP-Signalwege die Transformation von kardialen Fibroblasten (CF) zu Myofibroblasten (MyoCF), einem zellulären Phänotyp, der unter anderem mit der persistenten Fibrotisierung des erkrankten Herzgewebes in Verbindung gebracht wird. In CF führte eine Überexpression von PDE2 zu eine starken Abnahme der basalen und β2-AR-vermittelten cAMP-Synthese und war ausreichend, um in Abwesenheit exogener, pro-fibrotischer Stimuli die Transformation zum MyoCF zu induzieren. In Übereinstimmung zeigten funktionale Analysen mit künstlich hergestelltem Bindegewebe aus PDE2-überexprimierenden CF eine deutliche Zunahme der Gewebssteifigkeit. PDE2 übte keinen Einfluss auf basale oder durch das atriale NP generiertes cGMP aus und reduzierte nur partiell die NO-induzierte cGMP-Akkumulation. Interessanterweise waren beide Stimuli in der Lage, trotz niedriger cAMP-Spiegel die PDE2-induzierte CF-Transformation zum MyoCF zu verhindern und lassen daher eine Redundanz dieser beiden sonst so gegensätzlichen Signalwege vermuten. Zur Untersuchung von PDE2 in Kardiomyozyten wurde ein transgenes (TG) Mausmodell mit spezifischer kardialer Überexpression herangezogen. Die Basalcharakterisierung zeigte eine erniedrigte Herzfrequenz (HR) mit kompensatorisch erhöhter, basaler Kontraktionskraft, sowie eine verminderte Maximalantwort bezüglich der HR nach akuter β-AR Stimulation. Auf molekularer Ebene war dieser Phänotyp mit einer verminderten Phosphorylierung verschiedener β-AR Zielstrukturen wie Troponin I, Phospholamban und Ryanodinrezeptor-2 assoziiert. Langzeitstudien belegten, dass eine Überexpression von PDE2 keine pathologischen Konsequenzen hat, sondern im Gegenteil die durchschnittliche Lebensspanne der Tiere eher verlängerte. Erste Studien im Herzinsuffizienzmodel der transversalen Aortenkonstriktion (TAC) zeigten bisher eine beständig erniedrigte HR und verminderte Wanddicken bei allerdings vergleichbarer Abnahme der kardialen Kontraktionskraft. Trotz der klaren Befunde und neuen Erkenntnisse über die vielfältige Rolle der PDE2 im Herzen lässt sich bisher noch nicht klar belegen, ob eine zusätzliche Aktivierung von myokardialen PDE2 tatsächlich im Sinne einer intrazellulären β-AR-Blockade die Progression zur Herzinsuffizienz verlangsamen oder verhindern könnte. Weitere darauf aufbauende Untersuchungen, wie z.B. eine akut induzierbare Aktivierung bzw. Deaktivierung in experimentellen Herzinsuffizienzmodellen könnten den Weg für die Entwicklung klinisch anwendbarer Ansätze zur therapeutischen Modulation dieser viel versprechenden Zielstruktur ebnen.

Οι σύγχρονες στρατηγικές στην ανακάλυψη νέων βιοδραστικών ενώσεων στοχεύουν, κατά κύριο λόγο, σε καλά χαρακτηρισμένους μοριακούς στόχους με σκοπό την επαγωγή ή την αναστολή συγκεκριμένων βιολογικών δράσεων και τον περιορισμό ανεπιθύμητων παρενεργειών. Έναν ανάλογο μοριακό στόχο αποτελεί η διαλυτή γουανυλική κυκλάση (sGC), η οποία βρίσκεται στο επίκεντρο του ερευνητικού ενδιαφέροντος την τελευταία δεκαετία. Παρόλο που ανακαλύφθηκε περίπου πριν από τέσσερις δεκαετίες ως μέλος της οικογένειας των κυκλασών, η έρευνα που ακολούθησε την ανακάλυψή της δεν χαρακτηρίστηκε από την ίδια πρόοδο που χαρακτήρισε έναν άλλο εκπρόσωπο της ίδιας οικογένειας, την αδενυλική κυκλάση. Μερικοί από τους λόγους της περιορισμένης μελέτης της sGC συνοψίζονται στην χαμηλή ενδοκυτταρική της συγκέντρωση, που συνεπάγεται δυσκολίες στην απομόνωση, στον καθαρισμό και το χειρισμό της, και στην έλλειψη ενός αποτελεσματικού συστήματος έκφρασής της. Επιπρόσθετα, μόνο μετά την ανακάλυψη του μονοξειδίου του αζώτου (ΝΟ), την αναγνώρισή του ως σηματοδοτικό μόριο καθώς και την ταυτοποίησή του ως τον EDRF στα τέλη της δεκαετίας του ’80, πραγματοποιήθηκε ουσιαστική πρόοδος στον τομέα των βιολογικών δράσεων της sGC. Ο καθαρισμός του ετεροδιμερούς ενζύμου και η ανακάλυψη ενός μορίου αίμης ανά διμερές έθεσε τη βάση για την αποσαφήνιση του μηχανισμού ενεργοποίησης της sGC από το ΝΟ και την ακόλουθη κατάλυση της μετατροπής του GTP σε κυκλικό GMP, αν και ο ακριβής μηχανισμός παραμένει μέχρι σήμερα άγνωστος. Η ταυτοποίηση, επομένως, του σηματοδοτικού μονοπατιού NO/sGC/cGMP αποτέλεσε τη βάση για την ανακάλυψη ότι φάρμακα όπως τα οργανικά νιτρώδη, που χρησιμοποιούνται στη θεραπεία της στηθάγχης για περισσότερο από έναν αιώνα, δρουν στην ουσία ως δότες μονοξειδίου του αζώτου μέσω βιομετατροπής τους και ασκούν τη δράση τους μέσω ενεργοποίησης της sGC και παραγωγής, ακολούθως, του δεύτερου αγγελιοφόρου cGMP. Tα παραπάνω δεδομένα οδήγησαν στον προσδιορισμό του ΝΟ ως σημαντικού σηματοδοτικού μορίου στη φυσιολογία καταρχήν του καρδιαγγειακού συστήματος. Η πρόοδος που ακολούθησε τη μελέτη του sGC-cGMP μονοπατιού αποκάλυψε την ευρεία παρουσία του ενζύμου σχεδόν σε όλα τα κύτταρα των θηλαστικών και την εμπλοκή του σε ουσιαστικές φυσιολογικές λειτουργίες, όπως την καρδιακή ομοιόσταση, τη χάλαση αγγειακών και μη λείων μυικών ινών, την αναστολή της συσσώρευσης/συγκόλλησης αιμοπεταλίων, την περιφερειακή και κεντρική νευροδιαβίβαση, την ανοσολογική απόκριση, τη μεταγωγή του οπτικού ερεθίσματος, ενώ παρουσιάζει και ρυθμιστική δράση στο γαστρεντερικό και ουρογεννητικό σύστημα. Ταυτόχρονα, απορρύθμιση του σηματοδοτικού μονοπατιού έχει διαπιστωθεί να εμπλέκεται στην φυσιολογία και την εξέλιξη συγκεκριμένων παθολογικών καταστάσεων όπως αρτηριακή και πνευμονική υπέρταση, καρδιακή ανεπάρκεια, αρτηριοσκλήρυνση και θρόμβωση, διαβήτης, νεφρική ίνωση και ανεπάρκεια, φλεγμονώδεις και νευροεκφυλιστικές παθήσεις, γαστρεντερικές διαταραχές, σήψη και καρκίνος, όπου εντοπίζεται είτε να υπερλειτουργεί είτε να υπολειτουργεί. Εξαιτίας της σπουδαιότητας και της σοβαρότητας των καρδιαγγειακών παθήσεων, όπου το σηματοδοτικό μονοπάτι υπολειτουργεί, το μεγαλύτερο κομμάτι της έρευνας που ακολούθησε σχετικά με την ανακάλυψη νέων βιοδραστικών ενώσεων εστίασε κυρίως στην ανάπτυξη αγωνιστών της sGC, παρέχοντας όχι μόνο ΝΟ-δότες με βελτιωμένες ιδιότητες αλλά, επιπρόσθετα, δύο νέες κατηγορίες ΝΟ-ανεξάρτητων ενεργοποιητών που περιλαμβάνουν διακριτές δομικά ενώσεις. Η πρόοδος, ωστόσο, στην αναστολή του ενζύμου κρίνεται εμφανώς υποδεέστερη, παρόλη την προφανή σπουδαιότητα ανάλογων ενώσεων στη διερεύνηση των cGMP-εξαρτώμενων βιολογικών δράσεων. Μεταξύ διάφορων ενώσεων, όπως οργανικών φωσφορικών ενώσεων, νουκλεοτιδικών αναλόγων, πορφυρινικών παραγώγων και άλλων, ο περισσότερο ισχυρός και εκλεκτικός αναστολέας που έχει αναφερθεί μέχρι σήμερα είναι το ODQ, το οποίο θεωρείται ότι δρα μέσω οξείδωσης του σιδήρου της αίμης με αποτέλεσμα την απώλεια της ικανότητας ενεργοποίησης του ενζύμου από το ΝΟ. Το μειονέκτημα του ODQ, ωστόσο, να δρα in vivo και σε άλλες αιμοπρωτεΐνες περιορίζει την εφαρμογή του και καθιστά επιτακτική την ανάγκη ανακάλυψης νέων εκλεκτικών αναστολέων. Σε αυτά τα πλαίσια, στόχο της παρούσας μελέτης αποτελεί ο σχεδιασμός, η σύνθεση και η βιολογική αποτίμηση νέων πιθανών αναστολέων της sGC. Με βάση τη χημική δομή του ODQ, ως την ένωση-οδηγό, σχεδιάστηκαν νέα τρικυκλικά ανάλογα του βασικού σκελετού του ινδολίου και του διϋδροϊνδολίου. Η επιλογή του ινδολικού δακτυλίου έναντι του κινοξαλινικού σκελετού του ODQ πραγματοποιήθηκε με στόχο να διερευνηθεί κατά πόσο ο νέος σκελετός θα έχει επίδραση στη διατήρηση ή/και την ενίσχυση της ανασταλτικής δράσης έναντι της sGC. Επιπρόσθετα, η επιλογή του διϋδροϊνδολικού σκελετού αποσκοπούσε στη διερεύνηση της επίδρασης που μπορεί να έχουν μη επίπεδες ενώσεις έναντι του επίπεδου ODQ. Ταυτόχρονα, σχεδιάστηκε μία σειρά ετεροκυκλικών δακτυλίων προσδεδεμένων στους βασικούς σκελετούς, διαφορετικών από το δακτύλιο οξαδιαζολόνης του ODQ, ώστε να εξετασθούν πιθανές διαφορές της τελικής δράσης σε συνάρτηση με δομικές διαφορές των τρίτων δακτυλίων. Ακολούθως, πραγματοποιήθηκε διερεύνηση της συνθετικής μεθοδολογίας για την παραλαβή τόσο των τελικών προϊόντων όσο και σημαντικών ενδιάμεσων ενώσεων ως συνθετικών ενδιαμέσων για τη μελλοντική σύνθεση τροποποιημένων παραγώγων. Η σύνθεση των τελικών διϋδροϊνδολικών αναλόγων πραγματοποιήθηκε με κυκλοποίηση μέσω επίδρασης υδροξυλαμίνης ή διάφορων υδραζινών σε α-υδροξυμεθυλεν-κετο- ενδιάμεσα παράγωγα, τα οποία παραλήφθηκαν από τα αντίστοιχα 4-κετο-τετραϋδροϊνδολικά παράγωγα με την επίδραση μυρμηκικού αιθυλεστέρα. Τα αντίστοιχα ινδολικά τρικυκλικά προϊόντα συντέθηκαν από τα διϋδροϊνδολικά υπό συνθήκες αφυδρογόνωσης με επίδραση DDQ. Εξαιτίας των πειραματικών συνθηκών που εφαρμόζονται για την παραλαβή των συνθετικών ενδιαμέσων, κρίθηκε απαραίτητη για την εξέλιξη του συνθετικού σχήματος η προστασία, προηγουμένως, του πυρρολικού αζώτου με την κατάλληλη ομάδα. Με στόχο τη διερεύνηση της επίδρασης που μπορεί να έχει στην τελική βιολογική δράση η παρουσία ελεύθερου –ΝΗ στον πυρρολικό δακτύλιο, κρίθηκε σκόπιμη η αποπροστασία των τελικών προϊόντων. Παρόλο που εξετάσθηκε ποικιλία προστατευτικών ομάδων και συνθηκών αποπροστασίας, τα διϋδροϊνδολικά τρικυκλικά προϊόντα αποδείχτηκαν ασταθή. Κατέστη εφικτό να απομακρυνθεί μόνο μία προστατευτική ομάδα, η SEM ομάδα, και μόνο από τα πλήρως αρωματοποιημένα ινδολικά προϊόντα, τα οποία απδείχτηκαν σταθερότερα. Σύμφωνα με την παραπάνω μεθοδολογία συντέθηκαν 24 τελικά προϊόντα, από τα οποία 14 είναι διϋδροϊνδολικά παράγωγα και 10 ινδολικά, ενώ από τα τελευταία τα τρία φέρουν ελεύθερο πυρρολικό –ΝΗ. Εκτός από τα τελικά προϊόντα κατέστη εφικτό να συντεθούν και σημαντικά συνθετικά ενδιάμεσα. Τα α-μεθοξυ και α-αιθοξυκαρβονυλο- 4- ή 7-οξοτετραϋδροϊνδολικά παράγωγα παραλήφθηκαν με εφαρμογή διαφορετικής πειραματικής μεθοδολογίας και συντέθηκαν, επιπρόσθετα, τα α-υδροξυμεθυλεν-4-οξο-τετραϋδροϊνδολικά παράγωγα. Τα προαναφερθέντα συνθετικά ενδιάμεσα δύνανται να οδηγήσουν σύμφωνα με διαφορετικά συνθετικά σχήματα σε νέα τρικυκλικά ανάλογα. Επιπλέον, διερευνήθηκαν οι πειραματικές συνθήκες κάθε σταδίου της μεθοδολογίας με σκοπό τη βελτιστοποίηση των τελικών αποδόσεων. Μερικά από τα τελικά τρικυκλικά ανάλογα, τα οποία δεν έφεραν υποκατάσταση στις θέσεις 2- και 3- του πυρρολικού δακτυλίου, προωθήθηκαν σε in vitro βιολογικές μελέτες. Εξετάστηκαν, καταρχήν, όσον αφορά την ικανότητά τους να αναστέλουν την επαγόμενη από νιτροπρωσσικό νάτριο δραστηριότητα της sGC σε δύο διαφορετικές συγκεντρώσεις, 1 μΜ και 100 μΜ. Όλες οι υπό μελέτη ενώσεις αποδείχτηκαν αναστολείς του ενζύμου και παρουσίασαν ισχυρότερη δράση, με μία εξαίρεση, στην υψηλότερη συγκέντρωση. Οι ενώσεις 74 και 81 αποδείχτηκαν οι ισχυρότεροι αναστολείς με δοσοεξαρτώμενη δράση. Η ένωση 81 εξετάστηκε στη συνέχεια όσον αφορά στην εκλεκτικότητά της για την sGC σε μία μελέτη που περιελάμβανε επαγόμενη από τον ANF παραγωγή cGMP. Από την τελευταία μελέτη αποδείχτηκε ότι η παραπάνω ένωση δεν αναστέλει τις pGCs, υποδεικνύοντας ότι πιθανότατα η νέα σειρά ενώσεων δεν επιδρά στα άλλα μέλη της οικογένειας των κυκλασών, τις pGCs και τις ACs, και παρέχοντας μια ένδειξη για πιθανό μηχανισμό δράσης παρόμοιο με αυτό του ODQ. Η τελευταία υπόθεση ενισχύεται και από το γεγονός ότι οι υπό μελέτη ενώσεις δεν μεταβάλλουν τη βασική δραστηριότητα της sGC. Τα αποτελέσματα των πρωταρχικών βιολογικών μελετών επαληθεύουν τον κύριο στόχο της παρούσας μελέτης που ήταν η σύνθεση νέων αναστολέων της sGC. Η επιλογή του ινδολικού δακτυλίου, ως τον βασικό σκελετό των νέων ενώσεων, επιβεβαιώνει την υπόθεση του αρχικού σχεδιασμού, παρέχοντας αναστολείς με νέα δομικά χαρακτηριστικά. Πρώιμες μελέτες δομής-δράσης επισημαίνουν τη βελτιωμένη δράση που παρουσιάζουν τα επίπεδα ινδολικά προϊόντα έναντι των μη επίπεδων διϋδροϊνδολικών, αν και δεν είναι ακόμη δυνατό να εξαχθούν ασφαλή συμπεράσματα σχετικά με την επίδραση της δομής του τρίτου ετεροκυκλικού δακτυλίου. Περαιτέρω βιολογική αποτίμηση των υπόλοιπων προϊόντων της μελέτης καθώς και σύνθεση νέων τροποποιημένων αναλόγων θα εξυπηρετήσει την εξαγωγή λεπτομερέστερων συμπερασμάτων από μελέτες χημικής δομής-βιολογικής δραστικότητας και, πιθανότατα, την ανακάλυψη νέας ένωσης-οδηγού στο πεδίο της αναστολής της sGC. Τα συμπεράσματα που προέκυψαν από την παρούσα μελέτη όσον αφορά στη χημική σύνθεση ενδιαμέσων και τελικών προϊόντων αλλά και στη βιολογική αποτίμηση επιλεγμένων τελικών ενώσεων μπορούν να αποδειχτούν χρήσιμο εργαλείο για το μελλοντικό σχεδιασμό, εφαρμογή και σύνθεση νέων ενώσεων με τα απαραίτητα χαρακτηριστικά για αποτελεσματική αναστολή της sGC. Ανάλογες ενώσεις είναι δυνατό να αποδειχτούν όχι μόνο χρήσιμα πειραματικά εργαλεία αλλά και βιοδραστικά μόρια με πιθανή κλινική εφαρμογή σε συγκεκριμένες παθολογικές καταστάσεις, όπου η sGC υπερλειτουργεί, όπως στη σήψη. / Modern strategies in the field of drug discovery and development of new therapeutic agents are aiming mostly at well-characterized molecular targets in order to stimulate or inhibit specific biological actions without undesirable side-effects. Soluble guanylate cyclase (sGC) is such a molecular target that has been, among others, intensively reviewed during the last decade. Although it has been discovered as a member of the cyclase family four decades ago, the research regarding the enzyme hasn’t followed the progress made with its sibling, the adenylate cyclase. Some of the reasons for this delayed progress are summarized in the difficulties concerning the isolation, purification and manipulation of the protein as a result of its low intracellular concentration and the lack of an efficient expression system. In addition, only after the discovery of nitric oxide (NO) as a signaling molecule and its recognition as the endothelium-derived relaxing factor (EDRF) at the late ’80s, was any significant progress made in the field of sGC biological activities. The purification of the heterodimeric enzyme and the discovery of the presence of one molecule of heme per dimer shed light in the mechanism of activation of sGC by NO and the consequent catalysis of the conversion of GTP into cyclic GMP, although no exact mechanism has been proved until today. The identification of the NO/sGC/cGMP signaling pathway served the discovery that drugs such organic nitrates, which have been used for the treatment of angina pectoris for over a century, practically act as NO-donors through bioconversion and exert their actions through activation of sGC and generation of the second messenger cGMP. The above results led to the establishment of NO as a significant signaling molecule in the physiology of cardiovascular system. The following progress concerning the study of the sGC-cGMP signaling cascade revealed a broad presence in almost all the mammalian cells and an implication in many substantial physiological processes, such as cardiac homeostasis, vascular and non-vascular smooth muscle relaxation, inhibition of platelet accumulation and coagulation, peripheral and central neurotransmission, immune response, transduction of light signals and regulating function in the gastrointestinal and urogenital systems. Apart from the physiological function, the NO/sGC/cGMP pathway is implicated in the pathophysiology of certain conditions as well, such as arterial and pulmonary hypertension, heart failure, arteriosclerosis and thrombosis, diabetes, kidney fibrosis and failure, inflammatory and neurodegenerative diseases, gastrointestinal disorders, sepsis and cancer, where it could be down- or up-regulated. Due to the significance and the severity of cardiovascular disorders where the expression or the activity of the pathway substituents is downregulated, most of the following drug discovery research focused on developing new agents that activate sGC, affording not only improved NO-donors but, additionally, two new categories of NO-independent activators, involving structurally diverse compounds. Regarding the inhibition of the enzyme, progress could not be considered as high, in spite of the tremendous utility of inhibitors in elucidating the cGMP-dependent biological processes. Among other compounds such as methylene blue, LY83583, organic phosphates, nucleotide derivatives, porphyrin analogues, the most potent and selective sGC inhibitor is ODQ, which is suggested to act through oxidation of the heme iron and consequent abolishment of the enzyme NO-activation ability. ODQ’s selectivity disadvantage, though, to inhibit in vivo not only the soluble guanylate cyclase but other hemoproteins as well, results in an urgent need for new selective sGC-inhibitors. According to the above facts, the aim of the present study involves the design, synthesis and biological evaluation of new possible sGC-inhibitors. Using the chemical structure of ODQ as a lead-compound, new tricyclic analogues were designed, bearing the basic indole or dihydroindole skeleton. The choice of the indole ring against the quinoxaline framework of ODQ aimed at the evaluation of how the new heterocyclic skeleton could contribute in the maintenance or the enhancement of the biological activity of the lead-compound. Moreover, the impact of non-flat molecules against ODQ was intended to be examined by the use of dihydroindole ring. Additionally, a series of different heterocyclic rings, diverse from the oxadiazolone ring of ODQ, fused to the basic skeletons were designated in order to study any possible influence based on the structure in inhibitory activity. Consequently, the synthetic methodologies for the synthesis of the final products were investigated, as well as the preparation of key molecules which could be used in the future as synthetic intermediates for the synthesis of additional modified derivatives. The synthesis of final dihydroindole tricyclic analogues was accomplished under cyclization conditions reacting hydroxylamine or various hydrazines with the α-hydroxymethylen-keto-intermediates, which were prepared from 4-keto-tetrahydroindole derivatives and ethyl formate. The corresponding indole final products were prepared from the dihydroindole ones under dehydrogenation conditions using DDQ. Due to the synthetic conditions applied for the synthesis of the key intermediate compounds, the substitution of the pyrrole nitrogen atom with a proper protecting group was considered crucial for the progress of the synthetic plan. Aiming to study the impact of the unsubstituted indole ring on the biological activity, the cleavage of the protecting group from the final products was necessary. Although, a number of protecting groups and of cleavage conditions were examined, the dihydroindole tricyclic products proved unstable. We only attempted to cleave one protecting group, the SEM group, and only in fully aromatized products, which proved more stable. 24 final products were synthesized following the aforementioned procedures, fourteen dihydroindole derivatives, ten indole derivatives among of which three bear unsubstituted pyrrole nitrogen atom. Apart from the final products, some significant synthetic intermediates were managed to be prepared. The α-methoxy- or α-ethyoxycarbonyl- 4- or 7-oxo-tetrahydroindole derivatives were synthesized according to different experimental procedures, as well as the a-hydroxymethylen-4-oxo-tetrahydroindole derivatives. The above synthetic intermediates may lead following different synthetic pathways to new tricyclic indole final compounds. In addition, the experimental conditions in each single step of the synthetic plan were investigated and determined on the purpose of improved yields. Some of the final tricyclic analogues, bearing no substituent at positions 2- and 3- of the pyrrole ring, were initially tested in in vitro biological assays. Firstly, they were tested concerning their inhibitory ability against SNP-induced sGC-activity using two different concentrations (1 μΜ and 100 μΜ). All the tested compounds proved to be sGC-inhibitors, being more potent at the higher concentration, except of one compound. Derivatives 74 and 81 were the most potent inhibitors and their inhibitory activity was dose-responded. Product 81 was then examined regarding its selectivity towards sGC by an in vitro assay involving ANF-induced cGMP production. The last assay proved that the above compound does not inhibit pGCs, suggesting that possibly the new series of compounds does not have any impact on either pGC or AC, determining a possible ODQ mechanism of action. Another conclusion from the biological assays that reinforces the last hypothesis is that the new compounds do not inhibit the basal sGC activity. The preliminary results of the biological evaluation confirm the main purpose of the present study which is the synthesis of new sGC-inhibitors. The choice of the indole ring as the basic skeleton of the new compounds verifies the initial design suggesting inhibitors with new structural characteristics. Preliminary SAR studies highlight the improved potency of flat fully aromatized indole derivatives in comparison with the non-flat dihydroindole ones, while there could not yet be safe conclusions derived regarding the structure of the third heterocyclic ring. Further biological evaluation of the rest of the products as well as the synthesis of new additional modified compounds will serve more detailed structure-activity relationship studies and possibly the development of a new lead-compound in the field of sGC-inhibition. Conclusions related with the chemical synthesis of the intermediate and final products, as well as conclusions regarding the biological evaluation of selected tricyclic derivatives might be proved useful for the future design, application and synthesis of new molecules with all the appropriate structural characteristics for effective sGC-inhibition. Such compounds not only could be proved useful experimental tools but possible pharmacological modulators as well in certain conditions, like sepsis, where sGC is overexpressed.

ARAUJO, João Ronielly Campelo. Efeito antidepressivo-símile da frutalina, lectina α-d-galactose ligante, isolada de sementes de Artocarpus incisa L., em camundongos. 2016. 79 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Anderson Silva Pereira (anderson.pereiraaa@gmail.com) on 2017-04-03T18:22:06Z No. of bitstreams: 1 2016_dis_jrcaraújo.pdf: 2086872 bytes, checksum: 3373789680d8de097182e323441225d7 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-04-04T22:36:31Z (GMT) No. of bitstreams: 1 2016_dis_jrcaraújo.pdf: 2086872 bytes, checksum: 3373789680d8de097182e323441225d7 (MD5) / Made available in DSpace on 2017-04-04T22:36:31Z (GMT). No. of bitstreams: 1 2016_dis_jrcaraújo.pdf: 2086872 bytes, checksum: 3373789680d8de097182e323441225d7 (MD5) Previous issue date: 2016 / Frutalin (FTL), an α-D-galactose-binding lectin isolated from breadfruit seeds (Artocarpus incisa L.), has a range biological activities, but has not been conclusively shown to act on CNS disorders. In this study we evaluated the effect of FTL on mouse behavior. Mice (n=6/group) were treated with FTL (0.25; 0.5 or 1 mg/kg; i.p.) or vehicle (NaCl 0.9 %;10 mL/kg; i.p.) and submitted to hole-board (HBT), elevated plus maze (PMT), open field (OFT), tail suspension (TST) and forced swimming (FST) tests. In a second set of experiments, yohimbine (1 mg/kg), ketamine (0.1 mg/kg), L-NAME (10 mg/kg) or methylene blue (10 mg/kg) were administered (i.p.) 30 min before FTL (0.5 mg/kg). In order to evaluate the subchronic effect of FTL, animals were injected with FTL (0.5 mg/kg) or vehicle for 7 days and submitted to FST on the first and last day of treatment. A molecular docking was conducted on the NOS enzyme and NMDA receptor. No changes were observed in HBT and OFT results. The smallest dose of FTL (0.25 mg/kg) was associated with an increase in the number of entries into closed arms in PMT (p<0.05). FTL reduced immobility in TST (0.25 and 0.5 mg/kg; p<0.05) and FST (0.25 mg/kg; p<0.05 and 0.5 mg/kg; p<0.01). In FST, the effect of FTL was dependent on carbohydrate interaction and protein structure integrity and it was reduced by ketamine (NMDA antagonist), L-NAME (non-selective NOS inhibitor) and methylene blue (soluble guanylyl cyclase inhibitor). The antidepressant-like effect remained after subchronic treatment. Matching the results of the experiment in vivo, the docking study indicated an interaction between FTL and NOS enzyme and NMDA receptor. In conclusion, FTL was found to have an antidepressant-like effect mediated by the NMDA receptor/NO/cGMP pathway. / Frutalina (FTL), uma lectina α-D-galactose ligante, obtida de sementes de Artocarpus incisa L., tem apresentado várias atividades biológicas, como ação na modulação de alvos moleculares e reversão de neurotoxicidade in vitro, porém, há pouca evidência de seu efeito sobre doenças no Sistema Nervoso Central (SNC). Diante disto, este estudo avaliou os efeitos neurocomportamentais da FTL em camundongos. Os camundongos (n = 6 / grupo) foram tratados com FTL (0,25; 0,5 ou 1 mg/kg; i.p.) ou Veículo (NaCl 0,9 % 10 mL/kg; i.p.; Controle) e submetidos aos testes da placa perfurada, labirinto em cruz elevado, campo aberto, suspensão pela cauda ou natação forçada. Num segundo conjunto de experimentos, a ioimbina (1 mg/kg), cetamina (0,1 mg/kg), L-NAME (10 mg/kg) ou azul de metileno (10 mg/kg) foram administrados (i.p.) 15 min antes da FTL (0,5 mg/kg) e os animais foram submetidos ao teste de natação forçada. Verificou-se também se o efeito de FTL no teste de natação forçada era dependente da integridade estrutural e capacidade de interação a carboidratos. A fim de avaliar o efeito subcrônico da FTL, os camundongos receberam FTL (0,5 mg/kg) ou veículo durante 7 dias e submetidos ao teste de natação forçada no primeiro e último dia de tratamento. Foi realizado docking molecular da FTL com NOS e receptor NMDA. Os resultados mostraram que não houve alterações neurocomportamentais dos camundongos nos testes de placa perfurada e campo aberto. FTL na dose mais baixa (0,25 mg/kg) aumentou o número de entradas nos braços fechados no teste do labirinto em cruz elevado, permitindo sugerir um possível efeito do tipo ansiogênico. FTL reduziu o tempo de imobilidade nos testes de suspensão pela cauda (0,25 e 0,5 mg/kg; p <0,05) e natação forçada (0,25 mg/kg, p <0,05 e 0,5 mg/kg; p <0,01) apresentando um efeito antidepressivo-símile. A redução da imobilidade provocada pela FTL foi prevenida pela Cetamina (antagonista de receptores NMDA), L-NAME (inibidor não-seletivo da NOS) e azul de metileno (inibidor da cGMP), mas não pela Ioimbina (antagonista α2-adrenérgico). A desnaturação da FTL, bem como a sua associação à galactose, também preveniu o efeito antidepressivo-símile da lectina. O efeito antidepressivo-símile da FTL permaneceu o mesmo após tratamento subcrônico (7 dias) e não houve alteração no peso dos animais. Corroborando com os resultados in vivo, os estudos de docking molecular demonstraram que a FTL interage com a enzima NOS e receptor NMDA. Nossos resultados demonstraram que a FTL possui efeito antidepressivo-símile mediado por receptores NMDA e via L-Arginina/NO/cGMP, além de ser dependente de sua integridade estrutural e capacidade de ligação a α-D-galactose.