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Site-directed mutagenesis of human cytochromes P450 family 1 and their interacting partners
Milichovský, Jan ; Martínek, Václav (advisor) ; Befekadu, Asfaw (referee) ; Souček, Pavel (referee)
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 b5 can 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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Theoretical study of enzymes related to carcinogenesis: DNA polymerase β and cytochromes P450
Jeřábek, Petr ; Martínek, Václav (advisor) ; Entlicher, Gustav (referee) ; Ettrich, Rüdiger (referee)
Present doctoral thesis contributed to understanding of mechanistic principles of two enzymes participating in the process of carcinogenesis; DNA polymerase (pol ) and cytochromes P450 (CYP). Pol is part of the DNA base-excision repair mechanism (BER). The primary role of pol in, the BER mechanism, is inserting a new nucleotide into a DNA strand according to Watson-Crick base pairing rules. Pol plays an important role in the process of carcinogenesis, approximately 30 % of human tumors express pol mutants. The ability of pol to discriminate between "right" and "wrong" nucleotide during the insertion process is called fidelity. We employed computational methods to elucidate molecular basis of the fidelity of pol . First, the relative free energy calculation method LRA was employed to compare differences in free energies between the "right" and "wrong" nucleotide during its insertion into DNA. The results indicated a better stabilization of transition-state of the nucleophilic substitution catalyzed by pol in the case of the "right" versus "wrong" nucleotide. This difference resulted in an 80-fold contribution to its fidelity. Further, computational methods FEP and LIE were used to examine how mutations effect fidelity of pol . Results were than correlated with experimental data...
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Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level
Moriová, Magdalena ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...
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A role of microRNAs in the regulation of hepatic drug-metabolizing enzymes
Sedlmajer, Štěpán ; Smutný, Tomáš (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Štěpán Sedlmajer Supervisor: PharmDr. Tomáš Smutný, Ph.D. Title of diploma thesis: A role of microRNAs in the regulation of hepatic drug-metabolizing enzymes The liver is the main biotransformation organ in the body. Cytochromes P450 and conjugation enzymes are essential enzymes in the metabolism of drugs and other xenobiotics. Recently, there is growing evidence of post-transcriptional regulation of these enzymes through microRNA molecules. MicroRNAs are epigenetic regulators of expression, which usually bind to fully or partially complementary sequences within the 3'UTR (3' untranslated) regions of mRNA molecules. MicroRNAs have a role in interindividual variability of expression and activity of metabolic enzymes. Understanding their role and mechanisms of action may contribute to the development of new drugs and tailored pharmacotherapy. The work summarizes current selected achievements in the research on regulation of mainly hepatic cytochromes P450 and conjugation enzymes through microRNAs. The impact of RNA editing, single nucleotide polymorphisms and pseudogenes is also described.
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Effect of pH and other factors on the metabolic conversion of tyrosine kinase inhibitors.
Čillíková, Olívia ; Indra, Radek (advisor) ; Čermáková, Michaela (referee)
Targeted anti-tumor therapy is a modern and effective approach to cancer treatment. This type of therapy includes several groups of anticancer drugs. The second most commonly used group of targeted chemotherapeutic substances are tyrosinkinase inhibitors. These are small- molecular agents that target specific signalling pathways of the tumor cell, thereby inhibiting it's growth, proliferation and angiogenesis. Representatives of this drug group include vandetanib and sunitinib. Vandetanib is primarily used for the treatment of medullary thyroid cancer and sunitinib has been approved for the treatment of gastrointestinal stromal tumor and advanced renal cell carcinoma. The diploma thesis focuses on the effect of pH and temperature on the in vitro metabolism of vandetanib and sunitinib using first-phase biotrasnformation enzymes. Differently premedicated and control rat liver microsomes were used in the experiments. Human and rat recombinant cytochromes P450 were used for comparison. The primary purpose of the experiments was to find the experimental pH and temperature optimum of the in vitro metabolism of vandetanib and sunitinib. These experiments also investigated the effect of pH and temperature on the drugs themselves and their stability. The effect of pH and temperature on the metabolism of the...
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Effect of the anticancer drug cabozantinib on cytochrome P450 activity
Slobodníková, Eva ; Dračínská, Helena (advisor) ; Václavíková, Radka (referee)
Cabozantinib is an anti-cancer drug used mainly for the treatment of thyroid and renal cell carcinoma. It is classified as a low molecular weight selective tyrosine kinase inhibitor. Tyrosine kinases play a key role in signal transduction and regulation of many cellular processes such as growth, differentiation, and proliferation. The changes in the tyrosine kinase pathways are associated with the formation and progression of tumors where their growth is uncontrolled. Tyrosine kinase inhibitors act on tyrosine kinase receptors, thereby preventing the spread of cancer cells and slowing down the progression of cancer. Because cabozantinib, like other clinically used drugs, is metabolized by cytochromes P450, adverse drug interactions may occur that result in altered pharmacokinetics of the administered drugs and a consequent decrease in the efficacy of these drugs. In this diploma thesis, the effect of cabozantinib on the activity of the main enzymes of phase I biotransformation of xenobiotics, cytochromes P450, was investigated in vitro. The effect on the activity of both rat and human cytochrome P450 isoforms involved in xenobiotic metabolism was studied. CYP isoforms were predominantly incubated with cabozantinib at two concentrations; 10 µM and a concentration corresponding to the substrate...
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Metabolism of the mitotic inhibitor BTB-1 by first phase biotransformation enzymes
Vančurová, Kateřina ; Indra, Radek (advisor) ; Martináková, Lenka (referee)
Cancer is still one of the most common deaths in the world and therefore new drugs are needed to be developed to stop or slow down this disease. Recently, there has been a huge expansion in drug development. Cytostatics that are still widely used include a group of mitotic inhibitors aimed at inhibiting mitosis. A representative of a mitotic inhibitor is the small newly discovered molecule BTB-1. This molecule mediates reversible inhibition of the molecular motor Kif18A, which plays an important role in cell division. In the first part of this presented bachelor thesis, a suitable method for the determination of BTB-1 using high performance liquid chromatography was developed and subsequently its sensitivity was verified. Furthermore, a suitable extracting reagent was found. In the second part of the bachelor thesis, the metabolism of BTB-1 was studied by the microsomal system of non-premedicated rats and rats premedicated with various cytochrome P450 inducers. Subsequently, the time dependence of BTB-1 conversion was studied by the microsomal system of premedicated rats. The effect of different concentrations of BTB-1 on its metabolism was also studied using the microsomal system of premedicated rats. Furthermore, the metabolism of this new molecule was studied using cytosolar systems isolated...
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Activity of cytochromes P450 1A1, 1A2 and 3A4 expressed in eukaryotic and prokaryotic systems
Indra, Radek ; Stiborová, Marie (advisor) ; Mizerovská, Jana (referee)
Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...
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Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
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Isolation and characterization of microsomal fraction of fungus Pleurotus ostreatus and its role in the degradation of 17α-ethinylestradiol
Valášková, Petra ; Černá, Věra (advisor) ; Hodek, Petr (referee)
A synthetic hormone 17α-ethinylestradiol (EE2) which is a component of hormonal contraception pills has been identified as a main component of the endocrine-disrupting compounds (EDc). EDc are substances that mimic natural hormones in their action. Recently their amount especially in the groundwater and the surface water has been increased, which results in a negative impact on the hormonal system especially of aquatic organisms. Since it is not easy to replace these substances from the environment by conventional techniques other possibilities of their biodegradation are examined. White rot fungi, which are able to degrade lignin in nature, have promising biodegradation abilities towards many pollutants. These fungi contain a wide range of non-specific extracellular and intracellular enzymes that play an important role in the degradation. This bachelor thesis was targeted on the study of a white rot fungus, Pleurotus ostreatus, and especially on the degradation potential of its intracellular enzymes in the biodegradation of EE2. Initially, the ability of fungi Pleurotus ostreatus to degrade EE2 in vivo was tested. During the 48 hour incubation there was replaced 95,5 % of EE2. However, the role of cytochromes P450 (CYPs) in a metabolism of EE2 was not confirmed in this experiment by reason that an...
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