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Adaptation of enzymatic assays for high-throughput screening with use of ECHO-MS
Kráľová, Zuzana ; Mertlíková Kaiserová, Helena (advisor) ; Šácha, Pavel (referee)
High throughput screening for potential inhibitors of enzyme purine nucleoside phosphorylase (PNP) was developed during the work on this thesis. Products of PNP-catalysed reaction, hypoxanthine and ribose-1-phosphate, were quantified using mass spectrometry. Advantages and disadvantages of an Echo® MS device were explored and compared to the previously used radioenzymatic method. The emphasis was put on assay robustness and automatization of the protocol. Acoustic droplet ejection technique (ADE) and contactless dispensing of chemicals were employed, using Echo® 550 and Certus Flex® devices. These allowed to reduce reaction volume from original 20 µl to 5 µl and perform the whole reaction in a 384-well plate, decreasing the costs and increasing the throughput. Using a developed Echo® MS method, the time of analysis was shortened from 8 minutes per sample for radiometric analysis to 0,2 minute per sample. Furthermore, enzyme assay parameters, such as PNP concentration, composition of reaction mixture, incubation time and reaction termination were optimized, following the rules of HTS. The method developed in this work was employed for PNP inhibitors screening and their IC50 determination. Moreover, the HTS method enabled screening of the whole library of IOCB in-house synthesized compounds (~ 8000)....
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Antioxidative effects of the analogues of nucleic acid components.
Procházková, Eliška ; Mertlíková Kaiserová, Helena (advisor) ; Zusková, Iva (referee)
Reactive oxygen species (ROS) naturally occur in the organisms where they play e.g. signaling and antimicrobial roles. The imbalance between ROS production and removal results in the oxidative stress. This condition is largely considered as unfavourable leading to the oxidative damage of the biomolecules (lipids, proteins, nucleic acids). The oxidative stress accompanies a number of civilization diseases and may also arise as a consequence of pharmacotherapy (side effects of some drugs). Modulation of the oxidative stress with the use of antioxidants remains a subject of intense research. The analogs of nucleic acid components represent a structurally diverse class of compounds with important pharmacological potential. While their antiviral and cytostatic activities have been described in detail, information on their potential antioxidant activity is missing. The aim of this study was to identify the antioxidant activity in a series of 126 analogs of nucleotides, nucleosides and nucleobases and to describe structural motives responsible for this activity. Several methodological approaches have been used to reach the goal: 1) determination of the ability of the compounds to scavenge ABTS radical in vitro (TEAC), 2) the influence of the compounds on lipid peroxidation and 3) their ability to...
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Biotransformation aspects on novel carbocyclic nucleoside analogs.
Rozumová, Nela ; Mertlíková Kaiserová, Helena (advisor) ; Rumlová, Michaela (referee)
Carbocyclic nucleoside analogs with norbornane moiety that have been synthesized at IOCB AS CR, represent new potential chemotherapeutic agents with significant activity against Coxsackieviruses. The main objective of this work was to study the metabolism and mechanism of action of the original analog carbocyclic nucleoside MS 254, which is characterized by its antiviral and cytostatic effects. The attention was partially paid also to the two structurally related substances (MS 255, MS 320). In this work, we determined cytotoxicity of these compounds in cell culture and the effect of MS 254 on the amount of total and oxidized glutathione, activity of glutathione-S-transferase (GST), glutathione reductase (GR) and the effect on cellular oxidative stress. The kinetics of the conjugation of MS 254 by human GST was also studied. It was found that of the three substances tested MS 255 was the most cytotoxic and MS 254 was the least cytotoxic compound. It was further found that MS 254 does not cause significant oxidative stress and that it increases the activity of GST and GR in a dose-dependent manner. Michaelis-Menten constant of the conjugation of MS 254 with the glutathione (main metabolic pathway) was determined in the milimolar range, indicating a relatively low affinity of MS 254 for GST.
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Antioxidative effects of the analogues of nucleic acid components.
Procházková, Eliška ; Mertlíková Kaiserová, Helena (advisor) ; Zusková, Iva (referee)
Reactive oxygen species (ROS) naturally occur in the organisms where they play e.g. signaling and antimicrobial roles. The imbalance between ROS production and removal results in the oxidative stress. This condition is largely considered as unfavourable leading to the oxidative damage of the biomolecules (lipids, proteins, nucleic acids). The oxidative stress accompanies a number of civilization diseases and may also arise as a consequence of pharmacotherapy (side effects of some drugs). Modulation of the oxidative stress with the use of antioxidants remains a subject of intense research. The analogs of nucleic acid components represent a structurally diverse class of compounds with important pharmacological potential. While their antiviral and cytostatic activities have been described in detail, information on their potential antioxidant activity is missing. The aim of this study was to identify the antioxidant activity in a series of 126 analogs of nucleotides, nucleosides and nucleobases and to describe structural motives responsible for this activity. Several methodological approaches have been used to reach the goal: 1) determination of the ability of the compounds to scavenge ABTS radical in vitro (TEAC), 2) the influence of the compounds on lipid peroxidation and 3) their ability to...
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