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Structural characterization of influenza A polymerase PA subunit domains in complex with novel inhibitors
Radilová, Kateřina ; Kožíšek, Milan (advisor) ; Rumlová, Michaela (referee) ; Obšil, Tomáš (referee)
Influenza RNA-dependent RNA polymerase is a heterotrimeric complex and has an essential role in the life cycle of the virus. It is responsible for viral replication and transcription. One of its subunits, the polymerase acidic protein, interacts with the PB1 subunit via a crucial protein- protein interaction at its C-terminal domain. This 310 helix-mediated intersubunit interaction is required for the whole heterotrimer assembly. The N-terminal domain carries the endonuclease active site with two manganese ions. Both domains are considered promising drug targets. Current strategies to fight the influenza virus are limited to seasonal vaccines, and there are only a few anti-influenza drugs targeting mostly other viral proteins. Many used antivirals are susceptible to rapid resistance mutations development or cause severe side effects. This thesis provides structural insights into the two domains of the PA subunit. The first part is devoted to the characterization and optimization of a PB1-derived minimal peptide interacting with the C-terminal domain. Results from this part may be considered as a starting point for the rational design of first-in-class anti-influenza inhibitors of the PA-PB1 protein-protein interaction. In the other half, we have explored the inhibitory potency of flavonoids and...
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Development of high-throughput screening assay for the identification of inhibitors targeting influenza A polymerase
Karlukova, Elena ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...
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Interdoménové a intradoménové interakce u motorové podjednotky EcoR124I: Výpočetní studie
SINHA, Dhiraj
EcoR124I is a Type I restrictionmodification (RM) enzyme and as such forms multifunctional pentameric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on the motor subunit HsdR. When non-methylated invading DNA is recognized by the complex, two HsdR endonuclease/motor subunits start to translocate dsDNA without strand separation activity up to thousands base pairs towards the stationary enzyme while consuming ~1 molecule of ATP per base pair advanced. Whenever translocation is stalled the HsdR subunits cleave the dsDNA nonspecifically far from recognition site. The X-ray crystal structure of HsdR of EcoR124I bound to ATP gave a first insight of structural/functional correlation in the HsdR subunit. The four domains within the subunit were found to be in a square planer arrangement. Computational modeling including molecular dynamics in combination with crystallography, point mutations, in vivo and in vitro assays reveals how interactions between these four domains contribute to ATP-dependent DNA translocation, DNA cleavage or inter-domain communication between the translocase and endonuclease activities.
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