This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

published_or_final_version / Pathology / Master / Master of Philosophy

乙酰膽鹼酯酶主要存在於神經肌肉接頭處和中樞神經系統的膽鹼能突觸處，是神經遞質傳遞過程中極其重要的膽鹼水解酶。研究表明，阿茲海默病人的大腦通常呈現出乙酰膽鹼酯酶的異常表達和分佈，並伴隨著β澱粉樣蛋白的沉澱。目前，乙酰膽鹼酯酶抑製劑是治療阿茲海默症的主要臨床藥物。 / 在本研究中，我們利用Bac-to-Bac 桿狀病毒表達系統分別使人類和雞泡魚的重組乙酰膽鹼酯酶基因在家蠶幼蟲裡得到了高效的表達。我們將乙酰膽鹼酯酶的cDNA序列克隆到pFastBac{U+2122} Dual質粒的多角體蛋白啟動子下游。為了易於監控蛋白表達水平，橙色熒光蛋白的cDNA序列也被克隆到同一個質粒的p10啟動子下游。此外，我們將多聚組氨酸標籤加在了乙酰膽鹼酯酶基因的碳端，從而使蛋白的純化效率得到了顯著提高。我們通過皮下注射含有乙酰膽鹼酯酶的重組bacmid對五齡期的家蠶幼蟲進行了病毒轉染。感染後約4-7天，重組乙酰膽鹼酯酶在蠶蟲內成功得到了表達。酶促反應動力學研究表明，重組乙酰膽鹼酯酶的活性與來自相同物種的天然乙酰膽鹼酯酶基本相似。這種高效率、低成本、高產量的蛋白表達方法可以為我們提供大量的重組乙酰膽鹼酯酶，用於體外篩選治療阿茲海默症的乙酰膽鹼酯酶抑製劑。 / 隨著對阿茲海默症分子學水平上的進一步了解，研究提出乙酰膽鹼酯酶可能通過外周陰離子位點誘導β澱粉樣多肽聚集, 從而形成澱粉樣纖維。因此，理想的乙酰膽鹼酯酶抑製劑應該既有抑制乙酰膽鹼酯酶的活性，又可以對抗β澱粉樣蛋白沉澱的毒性, 從而達到神經保護的作用。因此，我們採用AutoDock Vina軟件對ZINC數據庫內的天然化合物進行了兩輪虛擬篩選，篩選出的化合物理論上是可以同時作用於催化位點和外周陰離子位點。接下來，我們將對候選化合物進行體外驗證。 / Acetylcholinesterase (AChE: EC 3.1.1.7) is the acetylcholine-hydrolyzing enzyme that plays an essential role on cholinergic neurotransmission at the synapses of the brain and at the neuromuscular junctions. Abnormal expression and localization of AChE have been observed together with Aβ deposits in the brain of Alzheimer’s disease (AD) patient. Currently, AChE inhibitors are clinically used as drugs for AD treatment. / In this study, we demonstrated high-level expressions of functional recombinant human AChE and Tetraodon nigroviridis AChE using Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. The cDNA of AChE was cloned into the polyhedrin (PH) promoter of the plasmid pFastBac{U+2122} Dual. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP2) was cloned downstream to the p10 promoter of the same vector. We engineered a polyhistidine-tag (His-tag) tail to the C-terminal of each AChE gene to facilitate the purification. Transfection was carried out by subcutaneous injection of the recombinant bacmid DNA containing the AChE gene into the silkworm larvae of 5th instar. Approximately 4-7 days of post-infection, the recombinant AChE was expressed in the hemolymph of infected larvae. The kinetic studies showed that the biological activities of the recombinant AChEs were comparable to that of natural ones from other sources. This rapid, low-cost, and high yield production method could provide us sufficient amount of recombinant AChE for in vitro screening of AChE inhibitors for AD treatment. / Further advances in understanding the molecular basis of AD have proposed that AChE promote the assembly of Aβ peptide into amyloid fibrils through interaction at the peripheral anionic site of AChE. Consequently, new classes of AChE inhibitors are expected to be able to inhibit the active site of AChE and, at the same time, to protect neurons from Aβ toxicity. Therefore, we applied two rounds of virtual screening of ZINC database using AutoDock Vina to obtain new potential inhibitors which might be able to targeting both of the active and peripheral sites of AChE. The compounds with good performances in both of the two rounds of screening would be validated by the sequential in vitro tests. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Shuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-124). / Abstracts also in Chinese. / Acknowledgements --- p.I / Abstracts (English) --- p.II / Abstracts (Chinese) --- p.IV / Table of Contents --- p.VI / List of Abbreviations --- p.IX / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Acetylcholine mediated neutotransmission in nervous system --- p.1 / Chapter 1.2 --- Acetylcholineterase --- p.2 / Chapter 1.3 --- Comparison of AChE and BChE --- p.3 / Chapter 1.4 --- Molecular sturcture of AChE --- p.5 / Chapter 1.5 --- Molecular diversity of AChE --- p.7 / Chapter 1.5.1 --- Regulation at transcriptional level --- p.8 / Chapter 1.5.2 --- Regulation at post-transcriptional level --- p.11 / Chapter 1.5.3 --- Regulation at post-translational level --- p.12 / Chapter 1.6 --- Classic functions of AChE --- p.15 / Chapter 1.7 --- Non-classic functions of AChE --- p.18 / Chapter 1.8 --- Diseases associated with AChE --- p.19 / Chapter 1.8.1 --- Myasthenia gravis --- p.19 / Chapter 1.8.2 --- Alzheimer's disease --- p.20 / Chapter 1.8.2.1 --- Pathogenesis of AD --- p.20 / Chapter 1.8.2.2 --- Treatments for AD --- p.22 / Chapter 1.9 --- Silkworm larvae as biofactory for protein expression --- p.24 / Chapter 1.10 --- Traditional baculovirus expression system --- p.26 / Chapter 1.11 --- Bac-to-Bac baculovirus expression system --- p.29 / Chapter 1.12 --- Virtual screening with AutoDock Vina --- p.29 / Chapter 1.13 --- Project overview and the aim of study --- p.31 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.1.1 --- Chemicals and Reagents --- p.33 / Chapter 2.1.2 --- Primers --- p.35 / Chapter 2.1.3 --- Antibodies --- p.35 / Chapter 2.1.4 --- Silkworms --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Construction of the expression cassette --- p.36 / Chapter 2.2.1.1 --- Preparation of E.coli competent cells --- p.36 / Chapter 2.2.1.2 --- Transformation --- p.36 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.37 / Chapter 2.2.1.4 --- Gene clean --- p.37 / Chapter 2.2.1.5 --- Subcloning of target genes --- p.38 / Chapter 2.2.1.6 --- Plasmid DNA extraction --- p.40 / Chapter 2.2.1.7 --- Quantification of plasmid DNA by spectrophotometer --- p.41 / Chapter 2.2.1.8 --- Plasmid DNA sequencing --- p.41 / Chapter 2.2.2 --- Generation of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.1 --- Transposition --- p.42 / Chapter 2.2.2.2 --- White/Blue screening --- p.42 / Chapter 2.2.2.3 --- Extraction of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.4 --- Analysis of recombinant bacmid DNA by PCR --- p.44 / Chapter 2.2.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.1 --- Raising silkworm larvae --- p.45 / Chapter 2.2.3.2 --- Preparation of transfecting solution --- p.45 / Chapter 2.2.3.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.4 --- Collection of hemolymph after protein expression --- p.46 / Chapter 2.2.3.5 --- Oral infection of sikworm larvae --- p.46 / Chapter 2.2.4 --- Purification of AChE --- p.47 / Chapter 2.2.4.1 --- Nickel-chelating afinity chromatography --- p. 47 / Chapter 2.2.4.2 --- Determination of protein concenttration by BCA assay --- p.47 / Chapter 2.2.4.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.4.4 --- Western blot --- p.49 / Chapter 2.2.5 --- Kinetic analysis of AChE --- p.50 / Chapter 2.2.5.1 --- Ellman assay --- p.50 / Chapter 2.2.5.2 --- Curve fitting --- p.51 / Chapter 2.2.6 --- Virtual screening --- p.51 / Chapter Chapter 3 --- Expression of recombinant AChEs in silkworm larvae --- p.55 / Chapter 3.1 --- Construction of the expression cassette --- p.55 / Chapter 3.1.1 --- Human AChE and Tetraodon nigroviridis AChE --- p.55 / Chapter 3.1.2 --- Amplification of target genes from the parent vectors --- p.56 / Chapter 3.1.3 --- Insertion of target genes into pFastBac Dual --- p.58 / Chapter 3.2 --- Generation of recombinant bacmid DNA --- p.60 / Chapter 3.2.1 --- Phenotype verification --- p.60 / Chapter 3.2.2 --- PCR analysis of the recoombinant bacmid DNA --- p.64 / Chapter 3.3 --- Expression of AChE in silkworm larvae --- p.66 / Chapter 3.3.1 --- Raising silkworms --- p.66 / Chapter 3.3.2 --- High-level expression of AChE in silkworm larvae --- p.68 / Chapter 3.4 --- Oral infeciton --- p.72 / Chapter Chapter 4 --- Analysis of the recombinant AChEs --- p.73 / Chapter 4.1 --- Purification of recombinant AChEs by nickel-chelating affinity chromatography --- p.73 / Chapter 4.2 --- SDS-PAGE and western blot analysis of the recombinant AChEs --- p.76 / Chapter 4.3 --- Kinetic studies of recombinant AChEs --- p.79 / Chapter 4.4 --- Virtual screening --- p.84 / Chapter Chapter 5 --- Discussion and conclusion --- p.95 / Chapter 5.1 --- Demonstration of high-level expression of recombinant AChEs by Bac-to-Bac baculovirus expression system --- p.95 / Chapter 5.2 --- Oral infection of silkworm larvae --- p.98 / Chapter 5.3 --- Characterization of recombinant AChEs --- p.98 / Chapter 5.4 --- Discovery of new AChE inhibitors by virtual screening --- p.100 / Chapter 5.5 --- Future works --- p.101 / Chapter 5.6 --- Other applications --- p.102 / Chapter 5.7 --- Conclusion --- p.102 / References --- p.104 / Appendix I --- p.125 / Appendix II --- p.127 / Appendix III --- p.129

Evidence is growing that agrochemicals are playing a role in the decline of amphibians in California. An area of concern is the Sierra Nevada Mountains, where insecticides used in the Central Valley are aerially transported to amphibian habitats. I examined the effects of two of these insecticides, endosulfan and chlorpyrifos, in two experiments on anuran larvae. For the first experiment I exposed Sierra Mountain yellow-legged frog (Rana sierrae) larvae starting at Gosner stage 25 to each insecticide for 63 d to determine median lethal concentrations (LC50) and sublethal effects on growth and development. In the second experiment Sierran treefrog (Pseudacris regilla) larvae were exposed to chlorpyrifos and endosulfan individually and in combination from Gosner stage 25 through metamorphosis to evaluate the interaction between these insecticides. In the first experiment the endosulfan LC50 was 19.8 µg/L (95% confidence interval, 15.3-52.2 µg/L) and the chlorpyrifos LC50 was 595 µg/L (95% confidence interval could not be determined). Endosulfan concentrations greater than 8 µg/L reduced growth but had no effect on time to metamorphosis. No larvae exposed to chlorpyrifos reached metamorphosis. All larvae exposed to greater than 737 µg/L died before the end of the experiment. Growth was reduced above 325 µg/L and cholinesterase was depressed at 737 µg/L compared to controls. In the second experiment the interactive effects of the insecticides depended on concentration and exposure duration. Chlorpyrifos alone did not affect survival or body size after 30 d, even at concentrations greater than the previously reported LC50. Survival and body size decreased with increasing endosulfan concentrations. In combination, 137 µg/L chlorpyrifos inhibited the negative effects of endosulfan on growth and survival and the positive effects of endosulfan on cholinesterase.

The primary aim of the work described in this thesis is to establish a foundation for the applicability of a biochemical biomarker, cholinesterase (ChE) activity in food animal species, as an instrument for evaluating exposure to pollutants as well as predicting high-level effects on public health. Secondary aims are to increase the awareness of pesticide users of anti-ChE exposure, to decide whether poisoning episodes involve anti-ChE by measuring residual effects in tissues, and to identify sources of contamination in food animal tissues. The ChE are specialized carboxylic ester hydrolases that break down esters of choline. They are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). Both AChE and BChE activities were found to be higher in cattle than in sheep and higher in erythrocytes than in plasma and serum. The anticoagulant heparin significantly affects AChE activity in plasma compared with EDTA. Of the different tissue tested, the mean of ChE activities was found to be highest in tissue from the liver, followed by lung, muscle, kidney and heart for sheep and cattle. In pigs, the ChE activities tested higher in kidney, liver, lung, muscle and heart. The effect of freezing on ChE activities in liver and muscle tissues was significant inhibition after 6 months at -80 °C, whereas decreased after 3 months at -20 °C. A technique to improve the purification of AChE in sheep tissue was developed. BW284c51 strongly reduced acetylthiocholine iodide (AcTChI) and propionylthiocholine iodide (PrTChI) hydrolysis and slightly affected that of butyrylthiocholine iodide (BuTChI) in the liver, while iso-OMPA had no significant effect for muscle BuTChI of sheep and pigs. Histochemical study of liver tissue found AChE localised mainly in the cytoplasm of the cell lining in the sinusoids. The optimal pH values of AChE and BChE in liver and muscle ranged between 7.8 and 8.5. Both AChE and BChE activities increased when increase the time course and temperature. The half maximal inhibitory concentration (IC50) was found to be higher for carbaryl than dichlorvos (DDVP) and diazinon (DZN). Very little residual AChE activity was seen in the liver, but more was found in muscles. In general, the rate constants of inhibition (ki) values for liver and muscles were increased in different pHs according to the rank order of 8.5 > 7.5 > 6.5, while in plasma it was decreased in different temperatures as follows: 20 °C > 30 °C > 40 °C. The final experiments were carried out at the rate of spontaneous reactivation (ks) of inhibited AChE by DDVP and DZN from liver and muscle was found to be higher in sheep compared to cattle and pig, while the aging of phosphorylated AChE (ka) was found to be higher in cattle compared to sheep and pig. In addition, this study indicated that the developed bispyridinium symmetric (K048) oxime seems to be promising reactivated to DDVP-inhibited AChE for sheep and pigs while HI-6 was effective in cattle.

Although the physiological roles of BChE are not yet determined to date, the importance of this enzyme is continuously increasing as it was found to be associated with several disorders like diabetes mellitus type 2, cardiovascular diseases, obesity and especially with Alzheimer’s disease (AD). In consequence, for investigations of BChE’s pathological role in these diseases and to find new medication strategies, the development of selective and potent inhibitors is necessary. For this purpose, the current work progresses in five chapters on the exploration of the chemical, physical and biochemical properties of tetrahydroquinazoline based carbamates which were previously reported to be selective BChE inhibitors with potency in the low nanomolar range. 1) A Novel Way to Radiolabel Human Butyrylcholinesterase for PET through Irreversible Transfer of the Radiolabeled Moiety: PET-radiotracers represent an innovative tool to determine the distribution and the expression of a biological target in vivo. BChE lacks to a large degree of such tracers with a few exceptions. In this work, methods were developed to incorporate the radioisotopes 11C and 18F into the carbamate moiety of an tetrahydroquinazoline based inhibitor. In contrast to reversibly acting PET-probes, the described radiotracers were proven by kinetic studies to transfer the radioisotope covalently onto the active site of BChE, thus labeling the enzyme directly and permanently. 2) Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode: To investigate the role of the tetrahydroquinazoline carrier scaffold on BChE inhibition, carbamate based inhibitors were synthesized. These compounds were successively used to perform kinetic investigations to determine their inhibition mode. Based on these data, a plausible binding model was postulated explaining the influence of the tetrahydroquinazoline carrier scaffold for binding at BChE’s active site just before carbamate transfer takes place. Additionally, these compounds feature neuroprotective properties and prevent oxidative stress induced cell death in their carbamate form as well as after the release of the tetrahydroquinazoline carrier scaffold. 3) Dual Addressing of Butyrylcholinesterase by Targeting the Catalytic Active Site (CAS) and the Peripheral Anionic Site (PAS): Compounds which are dual-targeting the CAS and the PAS of BChE are the most potent and selective BChE inhibitors to date with inhibition values in the picomolar range. In this work, a strategy is described how to turn tetrahydroquinazoline based carbamates into dual binding BChE inhibitors. These inhibitors feature a carbamate moiety which is covalently transferred onto the CAS of BChE, and in addition provide a second pharmacophore connected via a linker to the carbamate moiety which is proposed to target the PAS. Preliminary results reveal a high tolerance of BChE towards different linker lengths without decrease in affinity. 4) Investigation into Selective Debenzylation and Ring Cleavage of Quinazoline based Heterocycles: The tetrahydroquinazoline system is well investigated in terms of its synthesis and its selective oxidation. To explore the reactivity of this system, a tetracyclic tetrahydroquinazoline was exposed to common reduction agents. These experiments revealed a high sensitivity of the tetrahydroquinazoline core towards several reduction conditions 5) Experimental and Theoretical Investigation into the Stability of Cyclic Aminals: Tetrahydroquinazolines are known to degrade in acidic media through hydrolysis of their aminal system; but literature is lacking of a systematic investigation into this behavior. Therefore, different tetrahydroquinazolines were synthesized and exposed to phosphate buffered systems with defined pH-values. A clear increase of the hydrolysis rate of the aminal system was determined in dependency of an increasing acidic media. Computational studies predicted and experimental studies proved that hydrolysis takes place in an acidic environment while the condensation of this system is preferred in neutral or basic aqueous media. / Obwohl die physiologische Funktion der BChE zum aktuellen Zeitpunkt noch nicht vollständig aufgeklärt ist, so ist die Bedeutung dieses Enzyms hinsichtlich seiner möglichen Involvierung bei Diabetes mellitus Typ 2, kardiovaskulären Erkrankungen, Übergewicht und der Alzheimer-Erkrankung stetig steigend. Die Entwicklung von selektiven und hochwirksamen Inhibitoren ist daher notwendig um die Rolle der BChE im pathologischen Verlauf dieser Erkrankungen beurteilen zu können und ggf. neue Therapiemöglichkeiten zu eröffnen. In der hier durchgeführten Arbeit wurde in fünf Kapiteln die chemischen, physikalischen und pharmakologischen Eigenschaften von Tetrahydrochinazolin basierten Carbamaten untersucht. 1) Eine neuartige Methode zur PET-Radiomarkierung der menschlichen BChE durch den irreversiblen Transfer eines radioaktiv markierten Carbamatrestes: PET-Radiopharmaka (auch Radiotracer genannt) werden häufig verwendet, um die Verteilung biologischer Zielmoleküle in vivo bestimmen zu können. In der hier präsentierten Arbeit wurden Methoden entwickelt, um die beiden PET-Radioisotope 11C und 18F in den Carbamatrest eines Tetrahydrochinazolin basierten Inhibitors zu integrieren. Im Gegensatz zu herkömmlichen, reversibel agierenden PET-Radiotracern konnte bei den hier beschriebenen Radiotracern mittels kinetischer Untersuchungen gezeigt werden, dass sie den radioaktiv markierten Rest kovalent auf die BChE übertragen können, sodass das Enzym direkt und kontinuierlich radioaktiv markiert wird. 2) Entwicklung hochselektiver Carbamat-basierter BChE Inhibitoren durch rationale Untersuchung ihres Bindemoduses: Um den Einflusses des Tetrahydrochinazolingerüstes zur Hemmung der BChE untersuchen zu können, wurden veschiedenartige Tetrahydrochinazolin basierete Inhibitoren synthetisiert. Der Bindemodus dieser Verbindungen wurde dabei eingehend mittels ihrer Inhibitionskinetik untersucht. Auf Grundlage der dabei erhaltenen Daten konnte mithilfe computergestützter Methoden ein Bindemodell entwickelt werden, welches den Einfluss des Tetrahydrochinazolingerüstes zur Bindung des gesamten Inhibitors in das aktive Zentrum der BChE qualitativ wiederspiegelt bevor die eigentliche Inhibition durch den Carbamattransfer auf das aktive Zentrum stattfindet. Zusätzlich konnte gezeigt werden, dass die hier synthetisierten Verbindungen neuroprotektive Eigenschaften aufweisen, indem sie oxidativem Stress entgegenwirken. 3) Duale Adressierung des katalytisch aktivem Zentrums (CAS) und der peripheren anionischen Bindestelle (PAS) der Butyrylcholinesterase: Verbindungen, welche sowohl die CAS als auch die PAS der BChE simultan adressieren, gehören zu den potentesten und selektivsten BChE Inhibitoren mit Inhibitionswerten im pikomolarem Bereich. In der hier vorliegenden Arbeit wurde eine Strategie entwickelt, wie Tetrahydrochinazolin basierte Inhibitoren so modifiziert werden müssen, damit diese ebenfalls als dual-aktive Inhibitoren wirksam werden. Diese Inhibitoren weisen eine Carbamatfunktionalität auf, welche kovalent auf die CAS der BChE übertragen wird, und besitzen darüber hinaus ein zweites Pharmakophor, welches über einen Linker mit dem Carbamatrest chemisch verknüpft ist und an die PAS bindet. 4) Untersuchungen zur selektiven Debenzylierung und Ringspaltung von Chinazolin basierten Heterozyklen: Das Tetrahydrochinazolinsystem ist in der Literatur ausgiebig hinsichtlich seiner Synthese und selektiven Oxidation beschrieben worden. Um den Einfluss reduktiver Bedingungen auf dieses System zu untersuchen, wurde ein tetrazyklisches Tetrahydrochinazolin gezielt mit verschiedenen Reduktionsmitteln umgesetzt. Informationen über die Reaktivität des Tetrahydrochinazolinsystems sind unumgänglich bei der Entwicklung neuer Verbindungen auf Grundlage dieses Systems, um Nebenreaktionen zu vermeiden. 5) Experimentelle und Theoretische Untersuchungen zur Stabilität zyklischer Aminale: Tetrahydrochinazoline weisen ein Aminalsystem auf, welches im sauren Milieu hydrolytisch gespalten werden kann. Um die Stabilität dieses Systems systematisch zu untersuchen, wurden Tetrahydrochinazoline synthetisiert und in einem wässrigen Phosphat-gepuffertem System mit definiertem pH-Wert inkubiert. Bei diesen Untersuchungen konnte ein klarer Zusammenhang zwischen einem sinkendem pH-Wert und einer beschleunigten Zersetzung der Testsubstanzen beobachtet werden. Außerdem konnte mittels quantenmechanischen Berechnungen und weiteren Experimenten gezeigt werden, dass diese Reaktion im alkalischen oder neutralem Milieu reversibel ist.

El compuesto PF9601N es un derivado de propargilamina y un potente inhibidor irreversible de la enzima monoamino oxidasa B (IMAO-B) el cual fue identificado por nuestro grupo tras una extensiva búsqueda de potenciales IMAOs. Además de su potente capacidad inhibidora, el PF9601N posee varias propiedades neuroprotectoras demostradas en varios modelos animales y celulares de la enfermedad de Parkinson (EP). Estos efectos, los cuales han sido relacionados con la presencia de la propargilamina en su estructura, están mediados por acciones en vías involucradas en la neurodegeneración observada en otras enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA). Así, para estudiar más en detalle las propiedades beneficiosas del PF9601N investigamos sus efectos en un modelo in vivo de excitotoxicidad, un mecanismo implicado en el daño neuronal observado en las enfermedades neurodegenerativas. El hallazgo de que el PF9601N era capaz de evitar el daño excitotóxico mediante la disminución de la liberación inducida de glutamato y aspartato, y el aumento de la liberación de taurina así como mediante la prevención de la activación glial y la apoptosis proporcionó un valor añadido a este compuesto para ser considerado en la terapia de estas enfermedades. El tratamiento actual para la EA se basa principalmente en el uso de inhibidores de las enzimas colinesterasas (IChEs). Sin embargo, estos fármacos no son capaces de disminuir la progresión de la enfermedad y sólo producen una mejora temporal de los síntomas. Actualmente está ampliamente aceptado que la EA es una enfermedad multifactorial. En este contexto, la aproximación farmacológica más novedosa, conocida como aproximación de los MTDL (de las siglas en inglés “ligandos dirigidos hacia múltiples dianas”), propone el uso de compuestos multifuncionales capaces de abrazar varias propiedades biológicas. Esta tesis se centra en el estudio de la relación estructura-actividad (REA) así como la evaluación biológica de varios compuestos híbridos especialmente diseñados y sintetizados para actuar sobre múltiples factores involucrados en la EA. Los compuestos híbridos combinan la porción de bencilpiperidina de Donepecilo, un anticolinesterásico ampliamente utilizado en el tratamiento de la enfermedad, con el grupo propargilamina o indolil propargilamina presente en PF9601N, con el objetivo de obtener un compuesto capaz de retener la capacidad inhibidora de MAO así como las propiedades neuroprotectoras y antiapoptóticas de PF9601N. El trabajo presentado en esta tesis demuestra que algunos de los compuestos híbridos son potentes IMAOs (rango nM) y moderadamente potentes IChEs (rango subM). De entre todos los compuestos evaluados, ASS234 resultó ser un potente inhibidor de la agregación del péptido β−amiloide (A) y fue capaz de ejercer una acción protectora frente a la toxicidad inducida por Aβ y H2O2 en células neuronales. En resumen, los datos presentados en esta tesis doctoral sugieren que el compuesto ASS234 es un compuesto multidiana muy prometedor que podría tener un papel modificador en la EA dada su demostrada capacidad de interactuar con varias dianas involucradas en la patogénesis de esta enfermedad. / PF9601N is a propargylamine-containing irreversible monoamine oxidase B inhibitor (MAOBI) previously identified by our group in an extensive screen of potential MAOIs. Besides its potent inhibitory capacity, it possesses several neuroprotective properties demonstrated in different animal and cellular models of Parkinson’s disease (PD). The beneficial effects of PF9601N, which have been related to the propargylamine group present in the molecule, are mediated through actions in pathways that are commonly involved in the neurodegeneration observed in other neurodegenerative disorders such as Alzheimer’s disease (AD), thus making this molecule a promising agent in the therapy of this disease as well. Thus, to study the beneficial properties of PF9601N in depth, we investigated its effects against an in vivo model of excitotoxicity, an important mechanism involved in the neuronal damage observed in neurodegenerative diseases. The finding that PF9601N was able to prevent the induced excitotoxic damage by decreasing the evoked release of excitatory neurotransmitters and decreasing the output of the inhibitory and neuroprotective taurine as well as preventing the induced glial activation and apoptosis gave more value to this compound to be considered in the therapy. The current treatment for AD is the use of cholinesterase inhibitors (ChEIs) although there is also a NMDA receptor antagonist. However, far from stopping the disease’s progression, these drugs only produce a temporary symptomatic benefit, thus highlighting an urgent need to provide real disease-modifying drugs. At present, the most accepted notion is that AD is a multifactorial disease caused by many different factors and thus drug therapy with multifunctional compounds, the so-called multi-target-directed ligand (MTDL) approach, embracing diverse biological properties will have noticeable advantages over individual-target drugs or cocktails of drugs. In this context, this thesis focuses on the structure-activity relationship (SAR) study and the biological evaluation of different hybrid compounds specifically designed and synthesised to target multiple factors involved in AD. The hybrid molecules combine the benzyl piperidine moiety of Donepezil, a commonly used anticholinesterasic for the treatment of AD, with the propargylamine or the indolyl propargylamine substructure of PF9601N, with the aim of retaining the MAO inhibitory capacity as well as the neuroprotective and antiapoptotic properties observed for this compound. The work presented in this thesis demonstrates that some hybrid compounds are potent MAOIs (nM range) and moderately potent ChEIs (submicroM range). Among them, ASS234 has also been shown to reduce Αβ fibrillogenesis, and to protect neuronal cells from A and H2O2 toxicity. Thus, this compound has proved to be able to block the Aβ-induced cell death in two ways: by preventing caspase cleavage and activation and blocking LDH release. Overall, the present data suggest ASS234 as a promising MTDL that may have a potential disease-modifying role in the treatment of AD since it is able to interact with diverse targets involved in the pathogenesis underlying AD.

Thesis (Ph.D)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmaceutics. Title from title-page of electronic thesis. Bibliography: leaves 249-255.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.