Charles University Pharmaceutical Faculty in Hradec Králové Department of Pharmaceutical Botany Candidate: Bc. Martin Cymbál, DiS. Supervisor: doc. Ing. Kateřina Macáková, Ph.D. Diploma thesis 2019/2020: Interactions of alkaloids with transition metals III., pp. 86. Copper is one of the important trace elements in the body. It regulates various enzymatic pathways. This work monitors the chelating and reducing activity of isochinoline alkaloids of the family Amaryllidaceae. The alkaloids of Amaryllidaceae have a large number of effects such as analgesic, narcotic, antiarrhythmic, antihypertensive, bronchodilatory, chemotherapeutic, antiparasitic, uteretonic, locally anesthetic, mydriatic and many other significant effects. An important representative is galanthamine hydrobromide, which is therapeutically used and is an inhibitor of acetylcholinesterase. Galanthamine type alkaloids (galanthamine, galanthamine hydrobromide, chlidanthine), lycorine type (lycorine, galanthine), haemanthamine type (haemanthnamine, vittatine) and montanine type (montanine) were studied. The methodology of the work was to determine the chelating and reducing activity with the use of the hematoxylin indicator and bathocuproindisulfonic acid, at different pH or in dimethylsulfoxide. Of the alkaloids studied, galanthamine and...

Le cuivre est un micronutriment essentiel qui participe à de nombreux processus biologiques. Cependant, le cuivre libre est toxique pour l’organisme parce qu’il catalyse une réaction de type Fenton formant des espèces réactives de l’oxygène. Par conséquent la concentration en cuivre est finement régulée dans tous les organismes vivants. Les maladies de Menkes et de Wilson sont dues à des dérèglements de l’homéostasie du cuivre qui se manifestent respectivement par une déficience ou une accumulation de cuivre dans l’organisme. La maladie de Wilson est traitée avec des chélateurs du cuivre, qui provoquent des effets secondaires importants chez certains patients.Mon projet de doctorat consiste en l’élaboration de trois familles de peptides qui contiennent des acides aminés cystéines et en l’étude de leurs complexes de Cu(I) pour déterminer s’ils sont des candidats adaptés pour le traitement de la maladie de Wilson. L'interaction de certains peptides avec les ions Hg(II) ou Zn(II) a également été étudiée. En effet, le Hg(II) est un cation métallique possédant des propriétés similaires au Cu(I) et donc souvent utilisé pour modéliser le Cu(I) qui est sensible à l'oxygène et se dismute dans l’eau. Le Zn(II) est quant à lui omniprésent dans les cellules et un compétiteur intracellulaire potentiel du Cu().Les séquences des peptides ont été choisies selon trois stratégies différentes. Dans la première, des séquences inspirées de la boucle de liaison du cuivre de la protéine bactérienne CueR (copper efflux regulator), contenant deux cystéines, ont été étudiées afin de bénéficier de la sélectivité et de la sensibilité de ce régulateur. Dans une deuxième approche, des peptides contenant trois cystéines dans les motifs CxCxxC et CxCxC ont été étudiés pour combiner les avantage des peptides (bonne internalisation dans les cellules hépatiques quand ils sont judicieusement fonctionnalisés) et des tripodes (très forte affinité pour le Cu(I)) de l’équipe CIBEST. Finalement, la pré-organisation a été exploitée dans un tétrapeptide rigide où les deux cysteines sont liées dans un coude β préformé.Les trois peptides modèles du régulateur CueR miment la capacité de la protéine à accueillir exclusivement un ion Cu(I) dans des conditions d'excès de ligand et une forte affinité et sélectivité par rapport au Zn(II). Ces caractéristiques sont avantageuses dans la perspective du développement de nouveaux chélateurs du Cu(I).Les peptides contenant trois cystéines s’avèrent trop flexibles pour contrôler la spéciation des complexes du Cu(I). Par ailleur, ces peptides sont bien adaptés pour une coordination efficace du Hg(II) par trois groupes thiolates. Les différences structurales n’ont qu’une influence modeste sur les stabilités des complexes. La différence dans la coordination des peptides vis-à-vis des deux ions mous Hg(II) et Cu(I) démontre que l'utilisation du Hg(II) comme ion modèle pour la coordination du Cu(I) avec des peptides ou des protéines riches en soufre dans des conditions physiologiques n’est pas toujours appropriée.La pré-organisation de la structure peptidique est un élément clé du contrôle de la spéciation du complexe Cu(I) et de l’affinité des ligands pour le Cu(I). Le peptide CDPPC forme uniquement le cluster Cu4P3 avec une grande stabilité et une bonne sélectivité Cu(I)/Zn(II). Au contraire, les données expérimentales avec le tétrapeptide plus flexible CPGC montrent la formation d’un mélange de complexes polymétalliques de Cu(I). Il est intéressant de noter que le peptide simple CDPPC est capable d’imiter la formation des clusters Cu(I)-thiolates identifiés dans de nombreuses protéines impliquées dans l’homéostasie du cuivre, comme Cox17 ou Ctr1. CDPPC est intéressant pour mettre au point un chélateur intracellulaire du Cu(I), et sa fonctionnalisation afin de pouvoir cibler les cellules hépatiques pour le traitement de maladie Wilson sera donc pertinente dans le futur. / The essential micronutrient copper participates in several biological processes, like respiration, iron homeostasis, antioxidant defense or pigment formation. However, excess of copper can promote ROS formation and thus induce oxidative damages. Therefore, intracellular copper concentration is under strict control. Menkes and Wilson’s diseases are genetic disorders causing impairment in copper homeostasis leading to copper deficiency or overload, respectively. Wilson’s disease is treated by chelation therapy, but the presently used drugs have several adverse side effects.The aim of my Ph.D. work consisted of the design of three groups of cysteine containing peptides and the characterization of their Cu(I) complexes to determine whether they are appropriate candidates for the treatment of Wilson’s disease.The peptides were designed following three different approaches. In a first strategy, we attempted to take advantage of the outstanding selectivity and sensitivity of the bacterial copper efflux regulator protein CueR by studying oligopeptides based on the metal binding motif of CueR involving two cysteine residues. Second, three-cysteine containing linear and cyclic peptides were designed with the aim of merging the better internalization of peptides by hepatocytes and the high Cu(I) affinity of tripods previously studied in the Delangle’s lab. Finally, the advantages of a highly preorganized peptide structure were exploited in a short, rigid tetrapeptide where two cysteines were linked by a turn motif (CDPPC). For comparative purposes studies were also performed with another, less rigid tetrapeptide ligand containing the PG unit as a turn inducing motif.The three CueR model peptides resemble the ability of the protein to exclusively accommodate one metal ion under ligand excess conditions. This, combined with the large affinity and high selectivity vs. Zn(II), are the features that are advantageous in the view of the development of new Cu(I) chelators.The three-cysteine-containing peptides proved to be too flexible to control the speciation and hereby leading to the formation of several species. On the other hand, they are well adapted for an efficient trithiolate coordination of the thiophilic cation Hg(II). Structural differences in the three-cysteine containing peptides have minor effect on the affinity of the ligands towards Cu(I) and Hg(II) ions. The striking difference in the behavior of the peptides towards the two soft metal ions demonstrate that the use of Hg(II) as a probe for Cu(I) coordination with sulfur-rich peptides or proteins in physiological conditions may not always be fully appropriate.Preorganization of the peptide structure is a key element in the control of Cu(I) complex speciation and in the affinity of the ligands for Cu(I).CdPPC forms a single Cu4P3 cluster with high stability and displays large selectivity for Cu(I) with respect to the ubiquitous Zn(II). In contrast, The CPGC-Cu(I) system is characterized by a more complicated complex formation. It is worth to note, that the simple CdPPC peptide is able to mimic the Cu(I)-thiolate cluster formation that are typical in proteins like Ctr1 or Cox17. CDPPC is an interesting simple peptide candidate to be targeted to the liver cells for the localized treatment of Cu overload in Wilson’s disease.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lukáš Konečný Supervisor: prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of Thesis: Preparation of a method for detection of the extent of chelation of calcium and magnesium ions Calcium and magnesium are biogenic elements belonging to the second group of the periodic table. Calcium is the fifth most abundant element in the human body. In addition to its important role in bone homeostasis, calcium is involved in other processes such as blood clotting, muscle contraction and regulation of nerve excitability Magnesium is the cofactor of many enzymatic reactions essential for a row of physiological functions. The aim of this work was to propose a novel screening method for determination of the degree of calcium and magnesium chelation. In the first step the UV-vis spectra of the selected indicator o-cresolphthalein complexone and its complexes with calcium and magnesium were measured. This was followed by verification of the linearity and the determination of the sensitivity of the method. Concurrently the stability of the indicator and its complexes with both ions was measured. In the last step, the suitability of the method was verified on a standard chelator ethylenediaminetetraacetic acid...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidat: Jana Faberová Supervisor: doc. Ing. Kateřina Macáková, Ph.D. Title of Thesis: Interaction of chalcones with transition metals I. Iron is a biogenic metal element with unpaired d-electrons that plays a key role in the survival of unicellular organisms, plants and mammals. The electron shell of the iron atom is able to easily bind and release electrons and thus contribute to the proper function of the organism. Iron is necessary to ensure the transport of respiratory gases, cell differentiation and the functioning of some specific enzymes. However, despite the clearly positive effect of iron on human health, inappropriately low or high level of iron in the body can cause the development many diseases. Chalcones are plant polyphenols belonging to the family of flavonoids, secondary metabolites of plants. The term chalcone generally refers to a chemical structure with an ,-unsaturated ketone system based on 1,3-diphenylprop-2-en-1-on. These substances have a wide spectrum of biological activities and in the right amount can be good for human health. The aim of this diploma thesis was to monitor the iron-chelating and iron-reducting activity of selected chalcones: isoliquiritigenin, licochalcon A,...

Charles University in Prague Faculty of Phramacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Martina Šulcová Supervisor: Assoc. Prof. Ing. Kateřina Macáková, Ph.D. Title of the diploma thesis: Interaction of chalcones with transition metals II. Copper is one of the essencial elements in human body which is involved in the functioning of many enzymes and metabolic pathways. Although it appears in trace amounts in the body, its functions are irreplaceable. However, the ion level must be kept in balance by the various mechanisms of the body. If there is an excess or deficiency of copper in the body, this condition can lead to the development of health issues. Chalcones are substances belonging to the group of flavonoids. Their polyphenolic structure does not have a closed ring C. Studies describe their anticancer, anti-inflammatory, antiviral, antidiabetic, antibacterial, antioxidant, immunosuppressive and other properties. Chosen substances in the group of dihydrochalcones (naringin dihydrochalcone, neohesperidin dihydrochalcone, phloretin, phlorizin) and chalcones (isoliquiritigenin, licochalcone A) were tested for their chelation and reduction activity of the copper ions in this diploma thesis. The activity was measured at different pH environments (7.5; 6.8; 5.5 and 4.5) and...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Sobolová Supervisor: Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Consultant: Marcel Hrubša, MS.c. Title of diploma thesis: The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase Inhibitors of alcohol dehydrogenase play a key role in the treatment of poisoning caused by methanol, ethanol, ethylene glycol, and their toxic metabolites. Such treatment can therefore be considered as life-saving. In clinical practice, a single substance, fomepizole, is used. This thesis is aimed at investigation of the effect of eleven experimental chelators from the 4- acylpyrazol-5-ones group on equine and yeast alcohol dehydrogenases, and the possible definition of structural features that would be the most suitable for interaction with this enzyme. Due to the ability of these substances to chelate zinc ions, it is believed that they could inhibit an enzyme that has a zinc molecule in the structure. The measurement itself is based on principle of increase in absorbance during the enzymatic reaction, which is caused by the formation of NADH. This measurement is relatively simple, efficient and, due to used kinetic method, also less susceptible to be affected by...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Karolína Pelnářová Supervisor: Prof. Přemysl Mladěnka, PharmD., Ph.D. Consultant: Václav Tvrdý, MSc.; Marcel Hrubša, MSc. Title of diploma thesis: Searching of effective cobalt chelators - 8-hydroxyquinolines Cobalt is one of the essential trace elements present in the human body. It forms a part of the organometallic complex of vitamin B12, which is essential for many physiological functions. Both overload and lack of cobalt in the body is associated with pathological conditions. Manifestations of deficiency can lead up to pernicious anemia or hypofunction of the thyroid gland. Cobalt intoxication can occur in an industrial environment, such as by cobalt metal dust during heavy metal processing, or by its release due to corrosion from orthopedic prostheses. Systemic toxicity is manifested by a number of endocrine, cardiovascular and neurological symptoms. The main aim of this diploma thesis was to find effective chelators of cobalt from the group of substances derived from 8-hydroxyquinoline. In vitro spectrophotometric measurement was used to determine the degree of chelation. The effect of chelation was also monitored ex vivo in rat erythrocytes. All tested chelators - 8-hydroxyquinoline,...

New Zealand albino rabbits exhibited calcified aortic plaques and maximum average serum cholesterol levels of 1200 mg percent after twenty-three weeks on an atherogenic diet (250 to 500 mg percent cholesterol in ten percent corn oil; 200,00 I.U. vitamin D3 per month). One month following termination of the atherogenic diet, rabbits were treated with disodium edetate (Na2EDTA, 50 mg/kg body weight) via the marginal ear vein, on alternating days for a total of twenty infusions each. Aortae were examined for tissue calcium both quantitatively (direct microcomplexometric analysis) and histologically six weeks after completion of EDTA chelation therapy.

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This work involves the combination of chelation and microwave extraction as a technique for extracting adsorbed metals in soil. It has been termed in this work, Chelate Assisted Microwave Extraction (CAME). Unlike other extraction methods, CAME is able to differentiate between anthropological and geological trace metals. The method has been applied to major sample matrices included three types of soils (Bertie Sandy Loam, Davidson Silty Clay, and Davidson Clayey Loam) as well as sea sand, alumina, and iron oxide. In order to investigate the effects of sorptive coatings on metal concentrations, a sample set was first treated with humic acids, which represented organic coated materials. Afterwards, copper (II) solutions were spiked to both untreated and treated matrices and the efficiencies of recoveries were studied. Metal determinations were performed by Inductively Coupled Plasma-Atomic Emission Spectrophotometry (ICP-AES). Initial studies of control (untreated soil) and long-term Cu amended (spiked) Bertie Sandy Loam (BSL) indicated that extraction efficiencies were method dependent. Ethylenediaminetetraacetic acid (EDTA) was a primary chelating agent used for method comparisons. Extracting solutions having a pH range from 7.8 to 10.1 did not affect the EDTA-microwave method, but the increase in pH enhanced Cu removal by the EDTA-shaking extraction method which was a conventional chelation using a mechanical shaker. With the EDTA-microwave method, complete extraction was achieved in a short period of time (20 minutes). Compared to the EDTA-shaking method using two sandy soils, the EDTA-microwave method proved to be more efficient in extracting sorbed metals in soils (5.9 vs. 4.6 µg/g for the control BSL and 37.5 vs. 27.8 µg/g for the amended BSL. FT-IR and C13 NMR were used to characterize extracted and commercially available humic acids. The latter was employed to prepare the humate coated materials. Iron oxide containing materials such as Davidson Silty Clay (DSC), Davidson Clayey Loam (DCL), and iron oxide (Fe2O3), showed extremely high adsorption capabilities for humic acids, while sea sand, Bertie Sandy Loam, and alumina (Al₂O₃) provided lower adsorption capabilities for humic acids. Two sets of uncoated and humate coated materials were used for Cu spiking procedures using 0.001 M Cu (II) at pH 5.3 for 24 hour-equilibration. Three original soils and alumina showed high adsorption capabilities for Cu (II). The presence of coated humate helped increase Cu adsorption capabilities of these soils, but diminished the ability of alumina to react with Cu (II) ions. For HA-uncoated matrices, iron oxide had a higher Cu adsorption capability than sea sand but less than real soils. Humate coating improved Cu adsorption capabilities of not only real soils but iron oxide and sea sand as well. Lastly, the developed method named Chelate Assisted Microwave Extraction (CAME) was further examined and optimized. Several samples were investigated for extraction efficiencies. A two-step extraction, applying 120 W for 5 min followed by 60 W for 10 min, was efficient in removing sorbed metals from different matrices regardless of sorptive oxide and organic components. Concentrations of chelating agents slightly affected percent metal removal; however, there was no significant difference in results obtained using EDTA or diethylenetriaminepentaacetic acid (DTPA). Recoveries of spiked metals (Cu and Cd) ranged from 96 to 107%. Using the untreated (control) Davidson Silty Clay and Bertie Sandy Loam, the comparison of other chelating solutions were made. Clearly, CAME was promising and proved to be a good alternative to classical shaking method for extracting adsorbed metals from soils. / Ph. D.