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Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Proteolytic systems of the blood fluke (Schistosoma mansoni).
Fajtová, Pavla ; Horn, Martin (advisor) ; Bařinka, Cyril (referee) ; Sojka, Daniel (referee)
Schistosomiasis is a serious parasitic disease caused by blood flukes of the genus Schistosoma. It is a global health problem with more than 200 million people infected and 750 million people at risk. Current therapy relies on a single drug, praziquantel, for which there are concerns of emerging drug resistance. Proteases of schistosoma are promising target molecules for the development of new therapeutic strategies against schistosomiasis. This work focuses on the comprehensive characterization of proteolytic systems of Schistosoma mansoni and determination of their role in the interaction with the human host. First, the major proteolytic activities secreted by individual developmental stages of schistosoma that parasitize the human body were classified using functional proteomics. This analysis demonstrated their complex and specific distribution with predominant serine and cysteine proteases and metalloproteases. Second, tegumental and digestive proteases, namely prolyl oligopeptidase and cathepsins B, C and D, were identified by chemical genomics as suitable target molecules for therapeutic intervention. Prolyl oligopeptidase was biochemically characterized using a recombinant protein, its effective inhibitors were developed as templates for antischistosomal drugs, and a biological role of the...
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Synthesis of Neuraminidase binders suitable for theranostics
Berenguer Albiñana, Carlos ; Machara, Aleš (advisor) ; Cibulka, Radek (referee) ; Soural, Miroslav (referee)
Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...
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Inhibitors of the eukaryotic translation initiation factor eIF4E1
Pospíšilová, Klára ; Pospíšek, Martin (advisor) ; Roithová, Adriana (referee)
Protein synthesis is one of the most important processes that take place in a cell. Thus there are various mechanisms in a cell that regulate it. If that regulation fails it may lead to serious pathologies. An example of this is an abnormal increase in the production of eukaryotic translation initiation factor 4E1 which occurs in some types of cancer including head and neck squamous cell carcinoma, colorectal cancer, cervical cancer or lung cancer. Enhanced availability of factor 4E1 enables transformed cells to undertake a more intense translation. The expression of individual proteins is not increased to the same extent though. The enhancement in the level of factor 4E1 has a more significant effect on oncogenic proteins. Malignant transformation caused by an incorrect regulation of factor 4E1 can be precluded by an application of compounds that impair the activity of factor 4E1. A summarization of 4E1 inhibitors is the subject of this work.
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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...
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Parasitic protease SmCB2 as a target for the treatment of schistosomiasis
Bakardjieva, Marina ; Mareš, Michael (advisor) ; Mikeš, Libor (referee)
Blood flukes of the genus Schistosoma are parasitic trematodes that cause schistosomiasis, a serious disease afflicting more than 240 million people. The proteolytic system of schistosomes is essential for their viability: it participates in important processes during host-parasite interactions such as food digestion, invasion and tissue migration. Thus, schistosomal proteases are promising molecular targets for therapeutic intervention in schistosomiasis treatment. The thesis focuses on the protease cathepsin B2 from S. mansoni (SmCB2) which has not been studied in detail so far in terms of biochemical properties and biological function. Recombinant SmCB2 was prepared using yeast and bacterial expression systems and was chromatographically purified. Using an in vitro activity assay, the first effective inhibitors of SmCB2 were identified which inhibited its proteolytic activity in submicromolar concentrations. Specific polyclonal antibodies against SmCB2 were prepared and used for immunomicroscopic localization of this protease on the surface of the parasite. ELISA analysis demonstrated that SmCB2 is a parasite antigen recognized by the host immune system in the mouse model of schistosomiasis. The thesis provides valuable information about SmCB2 as a potential target molecule for synthetic...
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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
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Nonclassical noncovalent interactions in proteins and their importance for design of novel specific viral enzyme inhibitors
Kříž, Kristian ; Lepšík, Martin (advisor) ; Novotný, Marian (referee) ; Kabeláč, Martin (referee)
Noncovalent interactions are vital for functioning of biological systems. For instance, they facilitate DNA base pairing or protein folding. Recently, in addition to classical noncovalent interactions such as hydrogen bond, nonclassical noncovalent interactions have been discovered. An example of these interactions is halogen bond belonging to the class of σ-hole interactions, the knowledge of which is already being useful for medical compound design. The aim of this work is to find out if the chalcogen bond, also a σ-hole interaction, plays a role in the binding of existing viral inhibitors, too. Following that, we are also interested whether or to what extent can these existing chalcogen bonds be optimized for a greater affinity of the inhibitor binding. Several protein-ligand crystal structures exhibiting geometrical properties favoring a chalcogen bond have been found in the PDB database. We examined the interaction energies and the interaction energy geometrical dependencies of model systems derived from these crystal structures by means of quantum chemical calculations. Further we have optimized their strength by a series of substitutions. We thus propose that chalcogen bond can become a player in rational design of inhibitors of viral enzymes and their protein target. Keywords: Noncovalent...
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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...
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