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Design and synthesis of novel type of prodrugs of acyclic nucleoside phosphonates based on ProTides
Markušová, Katarína ; Janeba, Zlatko (advisor) ; Kundrát, Ondřej (referee)
This diploma thesis focuses on the synthesis of novel prodrugs of tenofovir, one of the main components of medications against human immunodeficiency virus (HIV) infection. The design of the compounds is based on the concept of ProTides and takes inspiration from the previous work of Filip Kalčic. In this work, the aromatic ester on the phosphonate moiety, typical for the ProTides, is replaced by an aliphatic ester. This aliphatic moiety is represented by a derivative of the amino acid L-serine. All of the target compounds were synthesized in a one-pot manner from three key components (L-serine, L-alanine, and tenofovir), except for one, the synthesis of which remains unsuccessful. The prepared compounds exhibit single-digit micromolar to low nanomolar potency against HIV-1. The most active compound (as a mixture of epimers) exhibited similar anti-HIV-1 activity to the reference ProTide prodrug, tenofovir alafenamide. Most importantly, none of the synthesized ProTides showed cytotoxicity. This was one of the key properties of the compounds from Kalčic's project that we wanted to improve. The results of this thesis represent a stepping stone to a new category of ProTide prodrugs of phosphonates. Key words: prodrugs, acyclic nucleoside phosphonates, ProTides, tenofovir, serine, TAF
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Cellular mechanisms of TRPA1 channel regulation
Barvíková, Kristýna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee)
TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...
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Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
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Cellular mechanisms of TRPA1 channel regulation
Barvíková, Kristýna ; Vlachová, Viktorie (advisor) ; Hudeček, Jiří (referee)
TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela ; Doubravová, Linda (advisor) ; Lichá, Irena (referee) ; Petříčková, Kateřina (referee)
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
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Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
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