Iron is the most abundant transition metal found in living systems and plays a crucial role in DNA biosynthesis. To accommodate higher replication rates, cancer cells require higher amounts of iron compared to non-neoplastic counterparts. This higher demand for iron renders cancer cells susceptible to iron deprivation, and exposure to iron chelators leads to growth arrest and cell death. Iron chelation strategies employing a wide variety of iron-binding scaffolds are currently under investigation for use in cancer treatment. Although these chelation approaches are effective against several cancer cell types, their use is limited due to toxicity ascribed to indiscriminate metal sequestration and induction of oxidative stress. Prochelation strategies in which the chelating unit remains inactive until triggered by a disease-specific event are expected to increase the specificity of chelation-based therapeutics. Chapter 1 provides an overview of chelation and prochelation based therapies as well as disulfide-based approaches in the design of prodrugs. In Chapter 2, the reduction activation mechanism of disulfide-masked thiosemicarbazone prochelators is described. Whereas disulfide-masked prochelators do not bind iron, reduction of the disulfide bond upon cellular uptake produces active chelators that readily bind intracellular iron. These systems are not active extracellularly; rather, they target the intracellular labile iron pool. We found that the antiproliferative activity of these disulfide-masked prochelators is dependent on the intracellular redox environment, with enhanced toxicity in more reducing conditions. The iron complexes resulting from exposure of cultured cells to the chelation systems were detected intracellularly by electron paramagnetic resonance in intact frozen cells. The compounds in our first series do not engage in intracellular redox chemistry and do not cause oxidative stress. In Chapter 3, the synthesis and characterization of a larger series of disulfide-masked prochelators featuring several classes of tridentate ligands is described. We investigated the iron-binding efficacy of the corresponding chelators, their ability to induce oxidative stress and their cell-cycle effects. We found that these prochelator systems, regardless of the identity of the donor set of atoms, do not result in the intracellular generation of oxidative stress. We also found that treatment of cultured cancer cells with prochelators results in cell-cycle arrest at G1/0 in non-synchronized cells and G2/M in G2-synchronized cells. In addition, we found that all classes of prochelators exhibit antiproliferative effects likely through induction of apoptosis. In Chapter 4, the syntheses and biological evaluations of disulfide-masked prochelators that feature carbohydrate targeting units are described. The sugar conjugates present increased aqueous solubility, compete as effectively as D-glucose for transporter-mediated cellular uptake, and are 6 to 11-fold more selective towards colorectal cancer compared to an aglycone that does not contain a targeting unit. The design of more potent prochelator systems, as well as the design of systems with improved selectivity and aqueous solubility are discussed in Chapter 5.

v angličtině Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Pohanová Supervisor: Assoc. Prof.. Přemysl Mladěnka, Pharm.D., Ph.D. Title of Thesis: Chelation of ferrous ions by xanthen-3-one derivatives Iron is an essential element important for proper function of cells. Imbalance of iron levels can lead to serious diseases. Since there is no excretory mechanism, the homeostasis is regulated at the level of absorption in the intestine. The iron overload, which leads to tissue damage due to catalysis of the formation of free radicals, occur because of genetic disorders such as hemochromatosis or owing to frequent administration of transfusions. Rational therapy for iron overload is the administration of the chelators. The aim of this study was to evaluate the ability of derivatives of 2,6,7- trihydroxyxanthen-3-one (synthesized at the University of Sarajevo - Dr. Durić) to chelate iron in 4 (patho) physiologically relevant pH conditions. The ferrozine spectrophotometric method was used to determine the degree of chelation. Measurements showed dependency of the chelating effect on pH: lowering pH resulted in the decrease of the effect. At pH 7.5, most of the substances showed 100% ferrous ion chelation in the stoichiometric ratio of 1...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Bc. Lenka Loskotová Supervisor: doc. Ing. Kateřina Macáková, Ph.D. Title of Thesis: Interactions of alkaloids with transition metals IV. Copper is a biogenic trace element that participates in the proper function of an organism. It is part of a number of enzymes and participates in metabolic processes in the human body. The level of copper in the organism should be regulated to avoid its excess or deficit, as pathologies could occur. Alkaloids are natural nitrogenous substances of alkaline nature. We find many biological activities in them. The aim of this thesis was to measure copper-chelating and copper- reducing activity of isoquinoline alkaloids: corypalmine, thalictricavine, 8-oxoberberine, fumarilin, norisocorydine and laurotetanine. Alkaloid activity was measured at different pH environments (4.5; 5.5; 6.8 and 7.5) and in the DMSO environment by a spectrophotometric method using hematoxyline and acid disodium salt bathocuproindisulfonic. Based on the results, structure-effect relationships were derived. In experimental measurement, it was found that none of us tested alkaloids showed chelating activity. Reduction activity has been demonstrated in all test substances. The lowest activity...

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...

Myoglobin (Mb) oxidation, and the subsequent browning, is the primary basis for consumer rejection of fresh retail beef. Considerable effort has been directed by the industry towards the development of techniques that can enhance color stability. However, the underlying mechanism of Mb oxidation has been studied extensively, but is still not entirely understood. It is known that chelation of iron and copper delays Mb oxidation and browning, but a clear role for these metals has not been established in any current Mb oxidation mechanism. The objective of the current study was to examine the possibility that iron plays a more direct role in Mb oxidation, and that metal chelators such as milk mineral (MM) and sodium tripolyphosphate can inhibit this action. MM, a colloidal calcium phosphate of large molecular weight and undetermined structure, was demonstrated to be a high-affinity iron chelator. Non-heme iron was found to stimulate Mb oxidation even in the absence of lipid, showing for the first time that the role of ferrous iron was not limited to promoting lipid oxidation, but instead has a yet-to-be determined role as a pro-oxidant factor in Mb oxidation. Ferrous iron was found to promote Mb oxidation under standard atmospheric conditions, while in high oxygen systems this effect was not seen. Addition of catalase did not affect Mb oxidation. However, in iron-containing systems, catalase significantly slowed Mb oxidation, while MM addition completely reversed the stimulatory effect of added iron. Type I radical-quenching antioxidants were found to rapidly reduce ferric iron to the ferrous form. This strong reducing ability accounted for the pro-oxidant effects of rosmarinic acid and eugenol in the lipid-free Mb model system. In raw ground beef, Type I antioxidants were highly effective at preventing Mb oxidation in the presence of lipid. Of the Type II chelators examined, only MM was able to delay Mb oxidation as well as the Type I antioxidants, possibly because it is not as susceptible to enzymatic hydrolysis.

Studies were undertaken to investigate if gastric acidity and iron chelation to a meat component enhance nonheme iron absorption. Cereal meals, with and without added proteins, were gavaged into iron-deficient rats. The role of iron chelation was investigated by adding sodium phytate, an iron chelator implicated with decreased iron absorption, to the meals. The role of gastric acidity was investigated by treating the rats with cimetidine, which inhibits gastric acid production. In rats with normal acid production, beef, pork and chicken enhanced iron absorption when phytate had been added to the meals, suggesting a role for chelation in meat enhancement of iron absorption. However, the enhancement by beef and pork was insignificant in cimetidine-treated rats given the cereal + phytate meals, indicating that gastric acid production also plays a role in meat enhancement of iron absorption. Fish and egg white were sometimes inhibitory to iron absorption and, therefore, did not fit the pattern of enhancement demonstrated by beef, pork, and chicken. In a separate experiment, gastric acidity was not directly altered by the protein source included with cereal meals. No significant effects of the various proteins on iron absorption from cereal + phytate meals were observed in a final experiment involving iron-replete rats. In vitro iron solubilizing capacity of beef, pork, chicken, and egg white was positively correlated with enhanced iron absorption by iron-deficient rats. Studies were performed to 1) investigate if ferric iron bound in complex with iron-solubilizing meat components is absorbable, 2) compare the relative iron-solubilizing capacity of meats, and 3) investigate the physicochemical and compositional characteristics of the meat components responsible for the iron solubilizing capability of meat. Iron-solubilizing components of beef were isolated from pH 2 HCl homogenates into dialysis bags (MWCO, 6-8 K). Radiolabelled iron complexes were then generated using ferric iron and either the ILC (isolated low-molecular-weight components) from undigested beef or ascorbate. The bioavailabilities of radioiron in these complexes or as ferric iron were measured as radioiron absorption into the blood one hour after injection into ligated duodenal loops of rats. Iron absorption values were ascorbate-ferrous complexes > beef ILC-ferric complexes > ferric iron (p < .05). In separate experiments, ILC from 0.1 g of various dietary protein sources (beef, pork, chicken, fish, or egg white) were added to 400 μg ferric iron in pH 2 HCl, the pH raised to 7.2, and soluble iron determined in the supernatant after centrifugation at 2,500 g for 10 min. Iron solubilizing capabilities of ILC were pork > beef > chicken > fish > egg white (p < .05). In a final series of experiments, the compositional and physicochemical characteristics of the ILC from the various dietary proteins were investigated.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Liptáková Supervisor: RNDr. Pavlína Hašková, Ph.D. Title of master thesis: In vitro study of newly synthesized potential cardioprotective drugs Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are in an organism generated under normal or pathological conditions. There are antioxidant mechanisms, which protects the organism from their harmful effect. In case of imbalance between ROS/RNS production and antioxidant mechanisms, an oxidative stress is initiated. The oxidative stress is involved in the pathogenesis of many diseases, including cardiovascular desease. In consequence of higher presence of mitochondria and lower presence of antioxidants cardiomyocytes are more sensitive to the oxidative stress. Iron, by catalysing radical's reactions, significantly participates on formation and development of oxidative stress. Elimination of the free iron by iron chelators is one option how to prevent or moderate oxidative stress. The aim of this master theses was to study cardioprotective effect in presence of H2O2 and own toxicity of newly synthetized aroylhydrazone iron chelators (H21, H22, H23, H24, H25 and H26) on rat embryotic cardiomyoblasts H9c2. Protective and toxic...