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Inhibition of TET-1 protein by iron chelators
Antonyová, Veronika ; Jakubek, Milan (advisor) ; Mikula, Ivan (referee) ; Gumulec, Jaromír (referee)
EN The objective of this work was to study novel synthetic iron chelators and to study their potential use in inhibition of TET1 (ten-eleven translocation methylcytosine dioxygenase 1) protein. Epigenetic mechanisms, such as hydroxymethylation of DNA, are promising target in many serious pathologies, including oncological disorders. In the presented study, we intended to discover novel inhibitors by screening small libraries of heterocyclic molecules, such as pyrrolo[3,2-b]pyrrole derivatives with hydrazide (compound 1) or hydrazone (compound 2-6) iron-binding group and hydrazone-based iron chelators (compound 7-10). Within the scope of this study, we used various analytical and biochemical techniques for the purpose of characterization of novel cancer therapies based on TET1 protein inhibition. The absorbance and the complexation of tested compounds with Fe(II) ions was studied by UV-Vis spectroscopy. Inhibition of TET1 protein was studied by fluorometric assay based on ELISA and further supported by microscale thermoporesis and in silico docking. The cytotoxicity of compounds on cancer cell lines was measured by MTT assay and intracellular distribution was determined by live-cell imaging. This study presents a biochemical analysis of potential TET1 protein inhibitors and brings significant...
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Study and mechanism of new-generation heterocyclic cytostatic with an emphasis on IL-6 andIL-8
Talianová, Veronika ; Jakubek, Milan (advisor) ; Mikula, Ivan (referee) ; Babula, Petr (referee)
EN The purpose of this study was to screen for novel synthetic inhibitors of interleukins IL-6 and IL-8, which are promising targets for cancer therapy. Small molecules are an attractive approach to inhibit these signalling pathways, which are known to be important for tumour cell growth and survival. The study aimed to discover new inhibitors of IL-6 and IL-8 signalling pathways by screening a small library of chemical derivatives, including π-expanded naphthalimide derivatives, Tröger's base, and pentamethinium derivatives. The structures of the compounds were designed based on in silico studies focusing on blocking the IL-6 and IL-8 signalling pathways. The study involved the use of various analytical and bioanalytical methods for the in vitro analysis of new potential anticancer drugs. The compounds were tested for cytotoxicity and their effect on cell growth using an MTT proliferation assay. Their antitumor activity was observed in NU/NU mice. The invasiveness of the cells was monitored using the wound healing assay. Live cell imaging was used to detect the distribution of the substances at the intracellular level. Other methods such as ELISA, microscale thermophoresis, and in silico predictions have been used to determine the selectivity of inhibitors of the signalling pathways IL-6 and IL-8....
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Eukaryotic and prokaryotic nitric oxide synthase - structure-function studies
Mikula, Ivan ; Martásek, Pavel (advisor) ; Entlicher, Gustav (referee) ; Král, Vladimír (referee)
Nitric oxide (NO) is an important signaling molecule in organisms. It plays a role in wide spectrum of physiological and pathophysiological processes, including vasodilatation, neurotransmission and host defense. The gaseous molecule of NO is produced by oxidative reaction catalyzed by proteins from the family of nitric oxide synthases (NOSs). Three NOS isoforms were identified in mammals, endothelial (eNOS), neuronal (nNOS) and inducible or immunologic (iNOS). Some bacteria harbor genes coding for proteins homologous to the mammalian NOS oxygenase domain and showing NO-producing activity in vitro. NO generated by pathologic organisms such as B. anthracis and S. aureus is supposed to play a critical role in the pathophysiological processes during the infection. Comparative study of bacterial NOS-like proteins and mammalian NOSs confirmed their principal similarity, but also revealed differences in the interactions of distinct bacterial proteins and mammalian NOS isoforms with different analogs of substrate L-arginine and various ligands. On the basis of the kinetics measurement of NO-rebinding a second NO-binding site in the active center of NOS was predicted. Further, the regulation of NO dynamic and release from the protein by the active site Hbonding network connecting the heme, the substrate and BH4...
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Eukaryotic and prokaryotic nitric oxide synthase - structure-function studies
Mikula, Ivan ; Martásek, Pavel (advisor) ; Entlicher, Gustav (referee) ; Král, Vladimír (referee)
Nitric oxide (NO) is an important signaling molecule in organisms. It plays a role in wide spectrum of physiological and pathophysiological processes, including vasodilatation, neurotransmission and host defense. The gaseous molecule of NO is produced by oxidative reaction catalyzed by proteins from the family of nitric oxide synthases (NOSs). Three NOS isoforms were identified in mammals, endothelial (eNOS), neuronal (nNOS) and inducible or immunologic (iNOS). Some bacteria harbor genes coding for proteins homologous to the mammalian NOS oxygenase domain and showing NO-producing activity in vitro. NO generated by pathologic organisms such as B. anthracis and S. aureus is supposed to play a critical role in the pathophysiological processes during the infection. Comparative study of bacterial NOS-like proteins and mammalian NOSs confirmed their principal similarity, but also revealed differences in the interactions of distinct bacterial proteins and mammalian NOS isoforms with different analogs of substrate L-arginine and various ligands. On the basis of the kinetics measurement of NO-rebinding a second NO-binding site in the active center of NOS was predicted. Further, the regulation of NO dynamic and release from the protein by the active site Hbonding network connecting the heme, the substrate and BH4...
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