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Non-structural proteins of Zika and Dengue virus with enzyme activity
Krýsová, Eliška ; Konvalinka, Jan (advisor) ; Novotný, Marian (referee)
Zika and Dengue viruses codes their own enzymes which helps them in different stages of the replication cycle. NS3 a NS5 proteins and their cofactors play an essential role in flaviviral life cycle. Although their structure was already solved, many aspects of their function remain unclear. The main subject of this bachelor thesis is the role of these proteins in flaviviral life cycle, polyprotein cleavage, replication and protein-protein interaction. These enzymes keep many particular enzymatic activities such as protease, helicase, methyltrasferase and polymerase. They are both structurally and functionally separated, which is interesting regarding autoactivation and protein-protein interaction. Since Zika and Dengue infections remain a serious health care issue, it is necessary to understand the molecular mechanisms behind their replication. Keywords: protease, polymerase, Zika, Dengue, polyprotein processing, antiviral therapy
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Preparation of Influenza Neuraminidase and Polymerase Inhibitors
Zima, Václav
Influenza is an infectious disease caused by the influenza virus. This virus causes a severe viral infection that spreads easily from person to person in yearly pandemics. Vaccination is the most effective way to prevent the infection, however, due to the high rate in mutations of the virus, the vaccine needs to be often reformulated. Another option to combat influenza is based on administration of antiviral drugs. Clinical studies of isolated influenza strains ("avian flu" H5N1, 2004; "swine flu" H1N1, 2009) revealed resistance towards known influenza neuraminidase inhibitors (zanamivir, oseltamivir). The resistance is caused by structural changes close to the enzymatic site. This calls for the development of new neuraminidase inhibitors as well for development of inhibitors targeting different influenza enzymes. This Thesis is focused on design and synthesis of new inhibitors of influenza neuraminidase and RNA-dependent RNA polymerase, namely PA subunit and the assembly of PA-PB1 heterodimer enzymes (Scheme 1). Influenza neuraminidase inhibitors were prepared by C-5 derivatization of oseltamivir followed by subsequent extension of its structure with binders of 150-cavity. Binding potencies of new oseltamivir derivatives against two influenza strains were determined. The next part contributed to...
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Synthesis of peptidic inhibitors targeting PA-PB1 interface of influenza RNA polymerase
Palacková, Miroslava ; Machara, Aleš (advisor) ; Veselý, Jan (referee)
The submitted Thesis deals with preparation of a hexapeptides inhibiting protein-protein interaction of PA-PB1 subunits of influenza RNA polymerase. Crucial part of the Thesis represent modifications of particular small hexapeptide at its two "hot spots". It means at positions that significantly contribute to the binding of both subunits. These modifications resulted in preparation of two series of distinct hexapeptides. With regards to the fact that one designed hexapeptide contains unnatural and commercially unavailable amino acids this amino acid had to be prepared from simple building blocks. Apart from aforementioned work the Thesis also covers effort to prepared bicyclic peptide that contains sequences of peptidic inhibitor of protein-protein interactions and also cell-penetration peptide. Key words: synthesis, peptides, inhibitors, influenza, polymerase
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Preparation of Influenza Neuraminidase and Polymerase Inhibitors
Zima, Václav ; Machara, Aleš (advisor) ; Cibulka, Radek (referee) ; Soural, Miroslav (referee)
Influenza is an infectious disease caused by the influenza virus. This virus causes a severe viral infection that spreads easily from person to person in yearly pandemics. Vaccination is the most effective way to prevent the infection, however, due to the high rate in mutations of the virus, the vaccine needs to be often reformulated. Another option to combat influenza is based on administration of antiviral drugs. Clinical studies of isolated influenza strains ("avian flu" H5N1, 2004; "swine flu" H1N1, 2009) revealed resistance towards known influenza neuraminidase inhibitors (zanamivir, oseltamivir). The resistance is caused by structural changes close to the enzymatic site. This calls for the development of new neuraminidase inhibitors as well for development of inhibitors targeting different influenza enzymes. This Thesis is focused on design and synthesis of new inhibitors of influenza neuraminidase and RNA-dependent RNA polymerase, namely PA subunit and the assembly of PA-PB1 heterodimer enzymes (Scheme 1). Influenza neuraminidase inhibitors were prepared by C-5 derivatization of oseltamivir followed by subsequent extension of its structure with binders of 150-cavity. Binding potencies of new oseltamivir derivatives against two influenza strains were determined. The next part contributed to...
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Modified ribonucleotides as building blocks for enzymatic construction of functionalized RNA or as antiviral compounds
Milisavljević, Nemanja ; Hocek, Michal (advisor) ; Baszczyňski, Ondřej (referee) ; Krečmerová, Marcela (referee)
The aim of this thesis was to study the steric influence of the base-modified nucleoside triphosphates (NTPs) on the enzymatic incorporation into RNA, as well as to study their inhibitory effect on different viral RNA polymerases in vitro. Their parent nucleosides and prodrug derivatives were also prepared and their antiviral activity evaluated. In the first part of the thesis, NTPs bearing groups varying in size from small methyl and ethynyl substituents via medium-size phenyl and benzofuryl groups, up to large dibenzofuran ring were prepared. Aromatic substituents were installed via Suzuki coupling on iodinated triphosphates or, in the case of modified guanosines, by the phosphorylation of modified nucleosides. Methyl and ethynyl NTPs were prepared via Pd-catalyzed coupling with AlMe3 and Sonogashira coupling, respectively, followed by the phosphorylation of modified nucleoside. To examine their incorporation into RNA by T7 RNA polymerase, templates coding for 35mer RNA containing one, three or seven modifications were designed. Modified pyrimidine triphosphates worked well for all the sequences, while the biggest dibenzofuryl group was not accepted in the difficult sequence with seven modifications. In the case of AR TPs dibenzofuryl modification did not incorporate at all, while other...
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Synthesis of peptidic inhibitors targeting PA-PB1 interface of influenza RNA polymerase
Palacková, Miroslava ; Machara, Aleš (advisor) ; Veselý, Jan (referee)
The submitted Thesis deals with preparation of a hexapeptides inhibiting protein-protein interaction of PA-PB1 subunits of influenza RNA polymerase. Crucial part of the Thesis represent modifications of particular small hexapeptide at its two "hot spots". It means at positions that significantly contribute to the binding of both subunits. These modifications resulted in preparation of two series of distinct hexapeptides. With regards to the fact that one designed hexapeptide contains unnatural and commercially unavailable amino acids this amino acid had to be prepared from simple building blocks. Apart from aforementioned work the Thesis also covers effort to prepared bicyclic peptide that contains sequences of peptidic inhibitor of protein-protein interactions and also cell-penetration peptide. Key words: synthesis, peptides, inhibitors, influenza, polymerase
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Non-structural proteins of Zika and Dengue virus with enzyme activity
Krýsová, Eliška ; Konvalinka, Jan (advisor) ; Novotný, Marian (referee)
Zika and Dengue viruses codes their own enzymes which helps them in different stages of the replication cycle. NS3 a NS5 proteins and their cofactors play an essential role in flaviviral life cycle. Although their structure was already solved, many aspects of their function remain unclear. The main subject of this bachelor thesis is the role of these proteins in flaviviral life cycle, polyprotein cleavage, replication and protein-protein interaction. These enzymes keep many particular enzymatic activities such as protease, helicase, methyltrasferase and polymerase. They are both structurally and functionally separated, which is interesting regarding autoactivation and protein-protein interaction. Since Zika and Dengue infections remain a serious health care issue, it is necessary to understand the molecular mechanisms behind their replication. Keywords: protease, polymerase, Zika, Dengue, polyprotein processing, antiviral therapy
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