The majority of drugs are small organic molecules, so-called ligands, that influence biochemical processes by interacting with proteins. The understanding of how and why they interact and form complexes is therefore a key component for elucidating the mechanism of action of drugs. The research presented in this thesis is based on studies of acetylcholinesterase (AChE). AChE is an essential enzyme with the important function of terminating neurotransmission at cholinergic synapses. AChE is also the target of a range of biologically active molecules including drugs, pesticides, and poisons. Due to the molecular and the functional characteristics of the enzyme, it offers both challenges and possibilities for investigating protein-ligand interactions. In the thesis, complexes between AChE and drug-like ligands have been studied in detail by a combination of experimental techniques and theoretical methods. The studies provided insight into the non-covalent interactions formed between AChE and ligands, where non-classical CH∙∙∙Y hydrogen bonds (Y = O or arene) were found to be common and important. The non-classical hydrogen bonds were characterized by density functional theory calculations that revealed features that may provide unexplored possibilities in for example structure-based design. Moreover, the study of two enantiomeric inhibitors of AChE provided important insight into the structural basis of enthalpy-entropy compensation. As part of the research, available computational methods have been evaluated and new approaches have been developed. This resulted in a methodology that allowed detailed analysis of the AChE-ligand complexes. Moreover, the methodology also proved to be a useful tool in the refinement of X-ray crystallographic data. This was demonstrated by the determination of a prereaction conformation of the complex between the nerve-agent antidote HI-6 and AChE inhibited by the nerve agent sarin. The structure of the ternary complex constitutes an important contribution of relevance for the design of new and improved drugs for treatment of nerve-agent poisoning. The research presented in the thesis has contributed to the knowledge of AChE and also has implications for drug discovery and the understanding of biochemical processes in general.

Ethanol extracts of “jenipapo” (Genipa americana Linnaeus), “umbu" (Spondia tuberosa Arruda), “siriguela” (Spondia purpurea Linnaeus) and “mangaba” (Hancornia speciosa Gomes) were prepared from separate pulp, seeds and peel; except mangaba which are used pulp and peel. The investigation of antioxidant capacity of the ethanol extracts were carried out by the methods of determining the content of ascorbic acid and total phenolics; scavenging 2, 2 -diphenyl-1-picrilhydrazyl and 2, 2’- azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical, antioxidant capacity of reduction of iron and copper and lipid peroxidation using a biomimetic membrane system, and measurement enzymatic of catalase and superoxide dismutase. It was also analyzed the acetylcholinesterase inhibitory activity, cytotoxic effect on corneal epithelial cells of sheep, as well as phytochemicals assays and identification of phenolic compounds and organic acids present in the extracts by UPLC - MS. The highest values of total phenolic content were obtained with peel and seed extracts and to ascorbic acid in the seed of siriguela, showing better antioxidant activities of seeds and peel extracts of siriguela and umbu. Lipid peroxidation assays indicated that genipap pulp is a promising antioxidant. Genipap pulp and siriguela seed ethanol extracts presented an acetylcholinesterase inhibition zone similar to that of the positive control, carbachol. The investigation of phenols and organic acid contents revealed the presence of quercetin (48,38 to 3,88 μg.g-1), quinic acid (43,28 to 41,88 μg.g-1) and citric acid (3,78 to 0,43 μg.g-1) in several extracts and chlorogenic acid with the highest amount found in siriguela seeds (356,93 μg.g-1). These data suggest new uses for these foods as potential antioxidant supplements for the food, pharmaceutical and cosmetic industries. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A partir das cascas, polpas e sementes do jenipapo (Genipa americana Linnaeus), umbu (Spondia tuberosa Arruda), siriguela (Spondia purpurea Linnaeus) e mangaba (Hancornia speciosa Gomes) foram preparados extratos etanólicos, com exceção da mangaba onde o extrato foi preparado utilizando casca e polpa. Os extratos foram submetidos à investigação da capacidade antioxidante através dos métodos de determinação do conteúdo de ácido ascórbico e fenóis totais, sequestro do radical 2,2-difenil-1-picril-hidrazil e 2,2´- azinobis (3-etilbenzotiazolina-6-ácido sulfônico), capacidade antioxidante de redução do ferro e cobre, lipoperoxidação utilizando um sistema de membranas biomimético e mensuração enzimática da catalase e superóxido dismutase. Também foi avaliada a atividade inibitória da enzima acetilcolinesterase, o efeito citotóxico em células epiteliais da córnea de ovelhas, além da realização de ensaios fitoquímicos e a identificação de compostos fenólicos e ácidos orgânicos presentes nos extratos. Os maiores teores de fenóis totais foram obtidos nos extratos das cascas e sementes e no referente ao ácido ascórbico na semente da siriguela, apresentando melhor atividade antioxidante os extratos das sementes e cascas da siriguela e do umbu. Porém, no ensaio de peroxidação lipídica, o extrato etanólico da polpa de jenipapo demonstrou ser um antioxidante promissor. Os extratos etanólicos da polpa do jenipapo e da semente da siriguela apresentaram uma zona de inibição da enzima acetilcolinesterase semelhantes ao controle positivo, carbacol. Na investigação dos compostos fenólicos e ácidos orgânicos por UPLC-MS verificou-se a presença nos extratos de quercetina (48,38 a 3,88 μg.g-1), ácido quínico (43,28 a 41,88 μg.g-1), ácido cítrico (3,78 a 0,43 μg.g-1) em vários extratos. E ácido clorogênico, na semente da siriguela (356,93 μg.g-1). Os resultados obtidos desses extratos sugerem novas formas de utilização desses alimentos como potenciais suplementos antioxidantes na indústria alimentícia, farmacêutica e cosmética.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Sabina Tanková Supervisor: PharmDr. Daniela Hulcová, PhD. Title of diploma thesis: Alkaloids of the genus Narcissus and their biological activity. Key worlds: Narcissus pseudonarcissus L. cv. Dutch Master, Amaryllidaceae, alkaloids, AChE, BuChE, POP, GSK-3β, biological activity. Alkaloid extract obtained from bulbs of Narcissus pseudonarcissus L. cv. Dutch Master was extracted by ethanol and was purified by liquid-liquid extraction and fractionated by column chromatography to individual fractions. At the end, were obtained 11 pooled fractions, which were used to isolate pure alkaloids. The ND 3-5 / 7 fraction was processed by preparative thin layer chromatography followed by crystallization of pure substances. In total, 5 alkaloid substances of ST1D2, ST1D3, ST2A, ST2B1 and ST3C were obtained from this fraction in various amounts. These substances were determined by GC-MS analysis, NMR analysis and optical rotation. Subsequently, the obtained data were compared with the NIST library spectra and the literature. Isolated substances have been identified as caranine, O-ethyllycorenine, narwedine, pluviine and N-demethylhomolycorine. The alkaloids obtained in sufficient amounts were subsequently subjected to...

Puskásová Dominika: Alkaloids of the Amaryllidaceae family and their biological activity I. Diploma thesis 2019. Charles university in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy. The aim of this thesis was to isolate alkaloids from herbal extract, which was obtained from Narcissus pseudonarcissus 'Dutch Master' plant. The preparation and column chromatography of the extract were performed by PharmDr. Daniela Hulcová, Ph.D. as a part of her doctoral study. Using the preparative TLC method, 2 alkaloids marked as No.2.1 and 2.2.2 were isolated from the fraction No.4. Their structure was determined by using the NMR, GC-MS analysis and optical rotation. After comparing the data obtained with literature, the compounds were identified as (+)-homolycorine and (+)-masonine. Both homolycorine and masonine were subsequently subject to testing of inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyloligopeptidase (POP) and glycogensynthase kinase 3β (GSK-3β). The activity was expressed as IC50 and was compared to IC50 of the reference substances. Galanthamine (IC50 AChE = 1,7 ± 0,1 μM, IC50 BuChE = 42,3 ± 1,3 μM) and huperzine A (IC50 AChE = 0,033 ± 0,001 μM, IC50 BuChE > 500 μM) were used as standards to compare the inhibitory activity...

Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Pharmacognosy (16-16180) Author: Štefan Kosturko Supervisor: PharmDr. Marcela Šafratová, Ph.D. Advisor: PharmDr. Daniela Hulcová, Ph.D. Thesis title: Alkaloids of the family Amaryllidaceae and their biological activity II. Key words: Narcissus pseudonarcissus dutch master; bulbs; alkaloidal extracts; GC/MS analysis; biological activity; acetylcholinesterase; butyrylcholinesterase. The main aim of the thesis ‚,Alkaloids of the family Amaryllidaceae and their biological activity II.'' was the isolation of alkaloids, as a pure substances, in sufficient quantities to identify their structure and to test their biological aktivity in vitro. Alkaloids were separated from subfraction number 82 - 97 of weigh 2,3268 g. This subfraction was a part of the total plant extract, which was prepared by PharmDr. Daniela Hulcová Ph.D., as a part of her dissertation thesis and who also performed primary extraction and separation work. A total and concentrated alkaloid extract weighing 58 g, which included the aforementioned subfraction, was obtained. The already mentioned alkaloid subfraction, was divided by preparative thin-layer chromatography into five separates, which were subjected to further phytochemical work, and five pure...

Růžička, L. Isolation of alkaloids from Geissospermum vellosii Allemão and study of their biological activity. Diploma thesis, Department of pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic, 2020. The aim of this diploma thesis are alkaloids from Geissospermum vellosii. This tree native in South America is commonly used for treatment of various diseases, including cognitive deficits in elder people1 . The study is based on previous research that was focused on inhibition of human cholinesterases and glycogensynthase 3 β(GSK-3 β), which can be used in treatment of Alzheimer disease. Incidence of this disease is rising up in the last decades and it represents a big burden for both health service and economy of developed countries2 . Isolation was carried out from crude crushed stembark. After extraction and agitation, 50.4 g of thick yellowish ether extract was obtained. This extract showed activity against cholinesterases (IC50 AChE = 15.19 ± 0.96 μg/ml and IC50 BuChE = 0.37 ± 0.049 μg/ml). Later, this extract was separated to 16 fractions by column chromatography. Fraction GV9 was chosen for additional research. Thin layer chromatography was carried out for purification and extraction of white crystalline alkaloid. Structure was determined by...

Eliška Emrichová: Isolation of alkaloids of the species Geissospermum vellosii Allemão and study of their biological activity IV. Diploma thesis 2020. Charles University, faculty of Pharmacy in Hradec Králové, Department of Pharmacognosy. Key words: Geissospermum vellosii, bark, alkaloidal extracts, isolation of alkaloids, GC/MS analysis, biological aktivity, acetylcholinesterase, butyrylcholinesterase. The aim of this study was to isolate at least one pure alkaloid from the extract of Geissospermum vellosii Alemão bark. The whole process involved bark processing, to obtain summary and alkaloid extract and subsequent column chromatography. GV-4, one of the 16 obtained fractions, was separated into 5 subfractions. The GV-4bsubfraction was used to isolate pure alkaloids, processed by preparative thin layer chromatography and crystallization of pure compound. The structure of pure compound was determined by using NMR, GC-MS analysis and optical rotation. This compound was identified as anhydropereirine and was tested its inhibitory activity against human cholinesterases, AChE and BuChE. The alkaloids GV-1-a, GV-8-3-B, GV-9-c were isolated in the course of further work on the extract. Their inhibitory activity against GSK-3β was tested as well as their possibility to cross the blood-brain-barieer with...

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La maladie d’Alzheimer (MA) est une maladie neurodégénérative complexe caractérisée par une perte progressive de la mémoire et de la cognition. C’est la première cause de démence chez le sujet âgé et affecte environs 4.6 millions de personnes par an, selon un rapport de l’association « Alzheimer’s disease International », le nombre de patients pourrait s’élever à 135.5 millions en 2050. Du fait de sa complexité, la MA reste incurable et seuls 4 médicaments aux vertus palliatives dont 3 visant à inhiber l’acétylcholinestérase (AChE) ont reçu une autorisation de mise sur le marché à ce jour. L’approche multi-cible parait particulièrement adaptée du fait du grand nombre de cibles potentielles de la pathologie, et du caractère multifactoriel de la maladie. Cette approche consiste à associer sur une seule molécule, plusieurs pharmacophores afin qu’ils puissent agir simultanément sur différentes cibles impliquées dans le processus neurodégénératif. Dans ce contexte, en parallèle de la resynthèse d’un ligand multi-cible conjugué alliant un inhibiteur d’AChE (IAChE) et un antioxydant, deux nouvelles familles de ligands multi-cibles conjuguées, combinant un IAChE et un agoniste des récepteurs nicotiniques α7 (α7 nAChR) ont été conçues et leur synthèse abordée. Dans le cas de la première famille, le fragment ivastigmine a été choisi pour sa capacité à inhiber de manière pseudo-irréversible l’AChE et a été conjugué à un motif quinuclidine, un puissant agoniste des α7 nAChRs impliqués dans la MA. En combinant ces deux fragments, il a été observé que les propriétés biologiques in vitro de chaque pharmacophore étaient améliorées. La structure de la seconde famille est basée sur le Donepezil, un IAChE réversible de plus forte affinité, combiné au même motif quinuclidine que dans la série précédente. Si des intermédiaires avancés ont été obtenus, un ou deux étapes restent à finaliser pour finaliser la synthèse de cette troisième famille de MTDL. / Alzheimer’s disease (AD) is a complex neurodegenerative disease characterised by a progressive loss of memory and cognition. Nowadays, 4.6 million new patients are identified every year and according to the “Alzheimer’s diseases International” association, the number of patients could reach 135.5 million in 2050. Due to its complexity, AD remains uncurable and only 4 palliative drugs, of which 3 are acetylcholinesterase (AChE) inhibitors (AChEI), have been approved by FDA to date. AD being a multifactorial illness, with many potential targets involved in the pathology, the MTDL approach seems promising. This strategy associates in one single molecule, different pharmacophores (at least) acting on different targets involved in this CNS-related disorder. In this context, in parallel with the upscaled synthesis of a conjugated MTDL combining an AChEI inhibitor and an antioxidant, two new families of conjugated MTDLs associating an AChEI and a α7 nicotinic receptor (α7 nAChR) agonist have been investigated. The structure of the first family was based on a Rivastigmine scaffold, known to be a pseudo-irreversible AChE inhibitor, and a quinuclidine fragment, a potent α7 nAChR agonist. By combining these two fragments, it was brought to light that the in vitro biological properties were improved on both targets. The second family was based on a donepezil fragment, a more potent AChEI, and the same quinuclidine fragment than in the first family. Advanced intermediates have been obtained, and two last steps remain to be achieved for the completion of this third MTDL series.

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