|
|
Methionine synthase as a potential therapeutic target
Kellovská, Kristýna ; Baszczyňski, Ondřej (advisor) ; Snášel, Jan (referee)
This thesis focuses on the enzyme methionine synthase (MS), which catalyzes methylation of homocysteine to produce methionine. Two main families of these enzymes are recognized in nature - cobalamin-dependent and cobalamin-independent. These two enzymes share no sequence homology, and they also use different catalytic mechanisms, substrates and cofactors. Cobalamin-dependent MS is found in humans, whereas cobalamin-independent MS is typical for plants and fungi. In humans, the enzyme provides a connection between folate and methionine cycle - two metabolic pathways which are crucial for example for DNA synthesis and S-adenosylmethionine-dependent biological methylations. Recently, the enzyme has been recognized as a potentially promising target for the development of chemotherapeutics and antifungal drugs, mainly based on its essentiality for the proliferation of cancer cells and both viability and virulence of pathogenic fungal species.
|
|
|
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...
|
|
|
Danio rerio as a model of serious human diseases
Hason, Martina ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
(ENGLISH) Over the last five decades, zebrafish (Danio rerio) has become a useful vertebrate model organism for the field of developmental biology and disease control. Using zebrafish in xenotransplantation studies is becoming more popular and progressed towards drug screening of anti-cancer drugs. Zebrafish are particularly suitable for high-throughput pre-clinical drug screening, due to the small size of embryos and the striking evolutionary conservation of cancer- related pathways between human and zebrafish. The fast, large-scale evaluation of the cancer- drug response in vivo could facilitate progress in personalized cancer therapy. Nevertheless, there is still a lack of methods which would allow for rapid and sensitive evaluation of tumor cell growth to facilitate high-throughput screening of drugs in vivo. In our bioluminescent zebrafish transplantation model, we proposed and validated a new screening platform for pre-clinical drug discovery in zebrafish embryos. In our experiments we used the NanoLuc luciferase, which enabled us to rapidly screen inhibitors of cancer growth in a sensitive and quantitative way with very low background compared to the conventional fluorescence signal. In our screen we evaluated the in vivo drug response of 180 kinase inhibitors in zebrafish embryos...
|
|
|
Derivatives of N-acetylglucosamine-1-phosphate as potential inhibitors of UDP-GlcNAc pyrophosphorylase
Černá, Lucie ; Baszczyňski, Ondřej (advisor) ; Veselý, Jan (referee)
This bachelor thesis deals with the preparation of phosphonate derivatives of N-acetyl- ᴅ-glucosamine-1-phosphate (GlcNAc-1-P), the substrate for UDP-GlcNAc pyrophospho- rylase (UAP1) enzyme, which is responsible for the chitin synthesis in fungal cell walls. Structural analogs of GlcNAc-1-P could potentially inhibit UAP1 and; therefore, their synthesis may enable the development of new antifungal drugs. This work is mainly focused on the synthesis of diethyl (2-acetamido-1,2-dideoxy-β-ᴅ-glucopyranosyl)phosphonate which was selected as a key model compound. The goal was to find the most suitable synthetic route leading to the synthesis of phosphonate and phosphinate GlcNAc-1-P analogues. Two synthetic methods were studied: 1) Michael addition of H-phosphonates to 2-nitroglucals; and 2) nucleophilic substitution of activated GlcNAc substrates (trichloroacetimidate and bromide) by phosphorus nucleophiles. Keywords: GlcNAc-1-P, UDP-GlcNAc pyrophosphorylase, antifungal, inhibitor
|
|
|
Development of inhibitors of amyloid fibril formation
Priss, Anastasiia ; Yushchenko, Dmytro (advisor) ; Schneider, Bohdan (referee) ; Hudeček, Jiří (referee)
α-Synuclein (α-Syn) is a small neuronal protein that is present in synapses, and, presumably, regulates the vesicle-mediated neurotransmitter release. Misfolding of α-Syn into amyloid fibrils is linked to the progression of synucleinopathies, including Parkinson's disease, the second most common neurodegenerative disorder. To date, no Parkinson's disease treatment that stops or retards neurodegeneration was established. Several direct and indirect approaches of influence on the α-Syn aggregation are extensively developed in search of potential drug candidates. Recently proposed inhibition of the α-Syn fibrils growth by blocking their ends is one of the most promising known strategies, as it opens an opportunity to design fibrillization inhibitors that act at very low concentrations. However, rational design of inhibitors of this type was not performed yet. The ultimate goal of this work was the development of highly efficient and specific inhibitors of α-Syn fibrillization. Therefore, careful structure-activity relationship analysis of the fibril end blocking inhibitors was performed and the affinity to fibril end was defined as the crucial factor for their inhibition activity. Using the recent data on the fibril structure, we designed inhibitors that are able to bind to the fibril ends with...
|
|
|
Inhibitors of proteolytic enzymes of Trematodes
Šteiger, Vladimír ; Kašný, Martin (advisor) ; Salát, Jiří (referee)
i Abstract Trematodes are important parasites possessing various localization in the bodies of invertebrate and vertebrate hosts, including human; therefore they are subject of long time intensive worldwide research. Trematodes developed various adaptations and strategies (some of them have also molecular background) enable them to survive in the host bodies. Trematodes produce large amount of different molecules, which are involved in various physiological processes. Inhibitors of proteolytic enzymes form a large group of biologically active compounds, e.g. they regulate the activity of peptidases or modulate host immune response. Many of these inhibitors are investigated as potential candidates in chemotherapeutic fight against trematodes. This thesis reviews the information concerning the natural inhibitors produced by trematodes and also synthetic inhibitors. Key words: Inhibitor, trematode, peptidase, serpin, cystatin i
|
|
|
Effect of cholinesterase inhibitors on monoaminergic system and energic metabolism
Kalinová, Tereza ; Mladěnka, Přemysl (advisor) ; Štěpánková, Šárka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Kalinová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Mentor: Assoc. Prof. Jana Hroudová, Pharm.D., Ph.D. Title of diploma thesis: Effect of cholinesterase inhibitors on monoaminergic system and energy metabolism Cholinesterase (ChE) inhibitors play an essential role in the treatment of Alzheimer's disease (AD). They effect positively cognitive, functional and behavior symptoms of AD. Up to date, donepezil, rivastigmine and galantamine represent the only ChE inhibitors approved for AD treatment. The first ChE inhibitor was tacrine, which was withdrawn from market due to its toxicity and adverse effects. Recently, novel tacrine and 7-methoxytacrine (7-MEOTA) derivatives were synthetized and extensively investigated to find less toxic compounds affecting pathological mechanisms associated with development of AD. There is less known about effects of these drugs on mitochondrial functions and cellular energy metabolism. The aim of this project is to examine in vitro effects of ChE inhibitors on energy metabolism and cellular respiration, specifically on mitochondrial electron transport chain complexes and an enzyme of the citric acid cycle - citrate synthase. Inhibitory effects...
|
|
|
Role of STAT3 signalling in oncogenesis and cancer therapy
Machalová, Veronika ; Hodný, Zdeněk (advisor) ; Brábek, Jan (referee)
STAT3 (Signal Transducer and Activator of Transcription 3) is considered to be one of the possible targets of cancer treatment. The ability of STAT3 constitutive activation to form tumors is a foundation of such theories. Additionally, constitutively activated STAT3 is present in many types of cancer with high occurrence, such as breast and prostate carcinoma. This protein is required in normal body cells as well. STAT3 is a transcription factor targeting many genes that are essential for the cell. STAT3 is activated by phosphorylation of its tyrosine residue and homodimerization. Proteins transcribed with help of STAT3 function in cell cycle progression, cell growth, replication, negative regulation of apoptosis, and other roles, typical for cancer. Moreover, STAT3 is modulating mitochondrial function and maintaining ROS production in mitochondria, but in form of transcriptionally inactive monomers. The purpose of this Thesis is to review known data about STAT3 in oncogenesis and by that, to show STAT3 has great potential to become the target of cancer treatment. This Thesis contains a short overview of known STAT3 inhibitors as well. Key words: Signal Transducer and Activator of Transcription 3 (STAT3), JAK/STAT3 pathway, constitutive activation, cancer, tumor, inhibitor, mitochondria, apoptosis
|
|
|
Vývoj chemických regulátorů drah mikroRNA a RNAi
Bruštíková, Kateřina ; Svoboda, Petr (advisor) ; Bařinka, Cyril (referee) ; Pospíšek, Martin (referee)
MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...
|
|
|
Experimental verification of in silico predicted protein binder to FOXO4 transcription factor and transcriptome analysis of bladder cancer
Tauš, Petr ; Drbal, Karel (advisor) ; Převorovský, Martin (referee)
This diploma thesis includes an experimental and a bioinformatic part. The two parts are linked together through the subject of transcription factors of 'forkhead box O' (FOXO) family. FOXO transcription factors have a key role in many cellular processes including cell cycle regulation, apoptosis and metabolism. For a long time, they have been considered strictly as the tumor-suppressors yet a growing number of evidence is pointing out to their pro-tumorigenic role. In consequence FOXO transcription factors are studied intensively as potential therapeutic targets in cancer. In the past decade, in silico prediction of protein-protein interactions has become popular in basic research as well as in drug development. Nonetheless, the predicted structures are still far from fitting to the expected behavior of the respective biomolecules. In the experimental part of this thesis, I verified the interaction of four in silico predicted protein binders based on naturally occurring PDZ domain with FOXO4 using microscale thermophoresis. Non-invasive bladder tumors represent a heterogeneous disease where reliable prediction of tumor aggressiveness is still lacking despite an intensive research. In the bioinformatic part of this thesis, I described the cellular composition of the tumor microenvironment and demonstrated...
|
guest ::
