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Oxidation of ellipticine by human cytochromes P450 expressed in prokaryotic and eukaryotic systems
Vejvodová, Lucie ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Ellipticine is an alkaloid with antitumor activity, whose mechanism of action is based on intercalation into DNA, inhibition of topoisomerase II and formation of covalent adducts with DNA, after its enzymatic activation by cytochromes P450 and/or peroxidases. Ellipticine is oxidized by cytochromes P450 to form up to five metabolites (7-hydroxy-, 9-hydroxy, 12- hydroxy-, 13-hydroxyellipticine and N2 -oxide ellipticine). 9-Hydroxy- and 7- hydroxyellipticine are considered to be detoxification metabolites, whereas 12-hydroxy-, 13- hydroxyellipticine and N2 -oxide of ellipticine are considered as activation metabolites, which are responsible for formation of covalent DNA adducts. The aim of this thesis was to examine the efficiency of human recombinant cytochromes P450 expressed in eukaryotic (SupersomesTM ) and two prokaryotic expression systems (Bactosomes) in oxidation of ellipticine. Cytochromes P450 expressed in prokaryotic systems differed in the amounts of "coexpressed" NADPH:CYP reductase. The resulting ellipticine metabolites were analyzed by HPLC. The results obtained in this thesis demonstrate that human cytochromes P450 2C9/2D6/2C19 expressed in prokaryotic or eukaryotic systems oxidize ellipticine to form up to four metabolites: 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and N2 -oxide...
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Interaction of Cytochromes P450 with Flavodoxin: a theoretical study
Culka, Martin ; Martínek, Václav (advisor) ; Chmelík, Josef (referee)
Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...
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Interaction of selected flavonoids with cytochromes P450 2B
Boštíková, Zdislava ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
In recent years, there has been increased emphasis on healthy lifestyle, which includes, among others, consumption of natural nutrients in a form of various food supplements. These compounds, usually of a flavonoid character, show different chemopreventive effects such as anti-oxidant or anti-carcinogenic. The excessive consumption of these compounds can also have a negative impact on human health. Flavonoids may for instance influence the content of biotransformation enzymes, cytochromes P450, in the cells and thus influence the metabolism of foreign compounds. Such an influence might, as a result, lead to a development of carcinogenesis or inefficacy of certain medications. The main focus of this bachelor thesis is to study capabilities of two flavonoid compounds (myricetin and dihydromyricetin) to induce and inhibit cytochromes P450 of 2B family. By the Western blot method it was found that neither of the investigated flavonoids increased expression of CYP2B1/2 in livers, myricetin, or in any part of small intestine. In the case of dihydromyricetin there was noticed an induction of cytochromes P450 2B in the middle part of small intestine. The inhibitory potency of flavonoids was examined in vitro as an inhibition of 7-pentoxyresorufin O-depentylation catalyzed by CYP2B. Values of IC50 8,8μM and...
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Effect of pH on vandetanib oxidation
Fedák, Michal ; Indra, Radek (advisor) ; Otáhalová, Barbora (referee)
Vandetanib is anticancer drug used mainly for targeted therapy of medullary thyroid carcinoma. It acts as inhibitor of tyrosine kinase and shows selectivity for vascular endothelial growth factor 2 (VEGFR-2) and epidermal growth factor (EGFR). It also inhibits rearranged during transfection (RET) tyrosine kinase activity. Vandetanib is metabolized by cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs) in organism of humans as well as experimental animals. CYPs oxidize vandetanib to N-desmethylvandetanib. FMOs are responsible for the formation of vandetanib N-oxide. This bachelor thesis studies effect of pH on vandetanib oxidation by CYPs a FMOs present in rat hepatic microsomes induced by different agents. Collected data show that in majority of series, optimal pH levels for oxidation of vandetanib by CYPs and FMOs are similar to a large extend. The highest amount of N- desmethylvandetanib was observed mostly at the pH 8,5. Vandetanib N-oxide was also produced in the highest quantity at the same level of pH in majority of series. Results suggest that N-desmethylvandetanib is formed at levels of pH which do not fit in interval of pH for optimal CYP activity. This finding is apparently due to a fact that presence of vandeanib in its neutral form, which is effectively oxidized by CYP,...
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Metabolism of cabozantinib by enzymes of first phase of biotransformation
Jurečka, Tomáš ; Indra, Radek (advisor) ; Kubíčková, Božena (referee)
Cabozantinib is an anticancer drug that inhibit tyrosine kinases which allow signal pathways important for growth and development of tumors. It is used for treatment of medullary thyroid cancer, hepatocellular carcinoma and kidney cancer. The major enzymes of the first phase of biotransformation that metabolize cabozantinib are cytochromes P450. In this thesis it was studied metabolism of cabozantinib and cytochromes P450 that participated on this metabolism. Hepatic microsomes of rat, mouse and rabbit were used for studying metabolism of cabozantinib in this thesis. It was also focused on the impact of particular isoforms of cytochromes P450 on metabolism of cabozantinib in rat microsomes. Time dependence of cabozantinib conversion in hepatic rat microsomes was also studied. Enzyme kinetics of metabolism of cabozantinib in hepatic rat microsomes, as well as impact of cytochromes P450 inhibitors on the metabolism were included. Metabolites were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. Formation of metabolites of cabozantinib increased over time to 30 minutes of incubation and with some others to 40 minutes of incubation. Up to five different metabolites were detected in experiments (M1, desmethyl cabozantinib, M3, monohydroxy cabozantinib and cabozantinib...
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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan
Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...
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Mechanism of carcinogenicity and nephrotoxicity of aristolochic acids
Bárta, František
Aristolochic acids (AA) are human carcinogens which have also very strong nephrotoxic properties. A mixture of AA is present in Aristolochiacae plant species. These plants were and still are used in traditional medicine in some countries, particularly in Asia. Aristolochic acids participate in development of two types of nephropathies. The first disease is designated as Aristolochic Acid Nephropathy (AAN), the second one is Balkan Endemic Nephropathy (BEN). Both nephropathies are associated with urothelial malignancies, which are caused by AA. One of the common features of ANN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumour development. One cause for these different responses may be individual differences in the activities and expression levels of the enzymes catalyzing the biotransformation of AAI, the major toxic component of AA contained in Aristolochia species. Detailed knowledge of enzymes which participate in metabolism of AAI may contribute to elucidation of inter-individual susceptibility to AAN, BEN and later urothelial malignancies. Aristolochic acid I is either oxidative detoxicated or reductive activated by biotransformation enzymes. Reductive bioactiovation of AAI leads to formation of covalent AA-DNA adducts in organism which result in producing of...
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The mechanism of action of anticancer drug ellipticin in target tissues of its effect
Mrízová, Iveta
Ellipticine is an alkaloid isolated from Apocynaceae plants exhibiting significant antitumor and anti-HIV activities. Cytochromes P450 (CYP) and peroxidases are the enzymes participating in metabolism of ellipticine. This process provides activation and detoxication metabolites of ellipticine. The CYP enzymes, which participate in oxidation of ellipticine in different tissues (liver, lung and kidney) of rat, a model organism simulating the fate of ellipticine in humans have already been identified. In this work, the effects of ellipticine on contents and catalytic activities of CYPs and other components of the mixed-function oxidase (MFO) system in this animal system were studied. For detection of contents of CYPs and other components of the MFO system, spectroscopic and electrochemical methods were used. To determine catalytic activities of CYPs and NADPH:cytochrome P450 reductase, reactions with specific substrates of these enzymes were utilized. The results found in this study demonstrate that expression and catalytic activity of CYP1A is induced by ellipticine in all of the tested organs (liver, kidney and lung) of rats treated with the drug. Moreover in liver, the cytochrome b5 expression is also induced. In addition, in this organ, expression and catalytic activity of CYP3A was increased by...
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Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450
Janoušková, Adéla ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Adéla Janoušková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450 Pharmacokinetic drug resistance often leads to failure of an anticancer therapy. One of the mechanisms is increased efflux of drugs from tumour cells, whereas some studies suggest that increased drug conversion to an inactive metabolite might be another contributing mechanism. The aim of this work was to define the possible role of CYP3A4 and CYP2C8 enzymes in the phenomenon of pharmacokinetic resistance and to investigate the possibility of its modulation by new targeted drugs. In the first part, we used the MTT proliferation method together with HepG2 cells stably transduced with particular human enzymes and demonstrated significant involvement of CYP3A4 in docetaxel resistance. In the following part, we examined the inhibitory effects of four selected tyrosine kinase inhibitors on the CYP3A4 activity in intact cells using a commercial kit. Cobimetinib and dabrafenib showed significant inhibitory activity, while osimertinib and brivanib did not. In the final part, we demonstrated the ability of the first two...
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Metabolism of inhibitors of tyrosine kinases, the drugs of new generation
Čillíková, Olívia ; Indra, Radek (advisor) ; Kubíčková, Božena (referee)
Cancer is the second major cause of death after heart-attack in the world. In recent years, research has focused on tyrosine kinase inhibitors (TKIs) as part of targeted chemotherapeutic treatment. Vandetanib is a TKI affecting epidermal growth factor receptor (EGFR), rearrangement during transfection (RET) and vascular endothelial growth factor receptor 2 (VEGFR2). It is primary used for treatment of medullary thyroid cancer. Vandetanib is biotransformed by cytochromes P450 and flavin monooxygenases in human organism. Cytochromes P450 (CYPs) oxidaze vandetanib to only one metabolite, N-desmethyl vandetanib, which exhibits similar efficiency as parental molecule. NADPH is the major cofactor of reaction cycle of CYPs. This bachelor thesis studies the effect of various types of cofactors and pH on oxidation of vandetanib by selected human recombinant cytochromes P450, namely CYP2C8 coexpressed with cyt b5, CYP2D6, CYP3A4 and CYP3A4 coexpressed with cyt b5. Here, we investigate the effect of cofactors NADPH, NADH and their mixture in a 1:1 ratio on the amount of N- desmethyl vandetanib formed during the biotransformation of vandetanib. The effect of pH on the oxidation of vandetanib by CYP 3A4 and CYP 3A4 + b5 was also analysed. We analysed the amount of the metabolite formed at the pH range 7 to 8.5...
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