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Synthesis of peptides as potential ligands of aspartate protease HIV and MAY1 and confirmation of their inhibitory activity
Klikarová, Ivana ; Šácha, Pavel (advisor) ; Žáková, Lenka (referee)
The lentivirus known as the human immunodeficiency virus (HIV) is transmitted through blood and body fluids, causing the destruction of CD4 lymphocytes and leading to opportunistic infections that define acquired immunodeficiency syndrome (AIDS). The urgent need for new antiretroviral drugs stems from concerns about the long-term toxicity of existing drugs, HIV-1 variants resistant to treatment, and frequent changes in patient treatment. Drug development is focused on inhibitors of two viral enzymes, reverse transcriptase and protease. Antiretroviral therapy uses protease inhibitors in combination with nucleoside analogs to effectively suppress viral replication, prolonging the lives of HIV-infected patients and reducing morbidity. Cryptococcus neoformans and cryptococcus gattii infections primarily affect the immunocompromised population and have high morbidity and mortality rates. Resistance to commonly used antifungals has been emerging, making it more difficult to treat these infections. Protease inhibitor components used in antiretroviral therapies have shown some clinical efficacy in these opportunistic infections, particularly in Major Aspartyl peptidase 1, an aspartate protease belonging to the same family of proteases as the HIV protease. To search for low molecular weight peptide ligands,...
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Preparation and characterization of vesiculin, a two-chain protein derived from IGF-2
Mrzílková, Karolína ; Žáková, Lenka (advisor) ; Pačesová, Andrea (referee)
Hormones of insulin family are one of the most studied groups of proteins due to their connection to large number of diseases including type two diabetes mellitus, tumorigenesis and some neurodegenerative diseases. The newest member of this superfamily is a two-chain analogue of IGF-2 called vesiculin. This peptide was originally isolated from secretory granules of mouse βTC6-F7 cell line and is currently believed to have a endocrine or paracrine effect on pancreatic β-cells. Its sequence homology with IGF-2 and structural similarity to insulin foreshadows vesiculin's potential metabolic, proliferation and growth properties and further secures its place as scientifically interesting and clinically lucrative topic. The insulin theme is corelated with insulin resistance, pathological state present primarily in early stages of development of type two diabetes mellitus. Vesiculin is able to stimulate glucose uptake and glycogen synthesis even in this condition which differentiates it form insulin and IGF-2 whose glucoregulatory activity is blunted in face of insulin resistance. Further research of vesiculin and insulin family receptor interaction, including dimeric tyrosinekinase receptor for IGF-1 and insulin, but also monomeric IGF-2 receptor, can provide more knowledge on character of this binding...
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Development of instrumentation and high-throughput screening methods for peptide ligand discovery and validation
Kryštůfek, Robin ; Konvalinka, Jan (advisor) ; Jiráček, Jiří (referee)
Peptides are used as synthetically available and easily derivatizable scaffold upon which it is possible to develop ligands targeting broad spectrum of biological targets. A time-tested approach to peptide binder identification is the preparation and screening of combinatorial libraries. Bypassing of this complicated procedure is possible by using biological systems for presentation, identification and selection of peptides based on the principle of in vitro evolution - i.e. display techniques. There are two complementary automated solutions for peptide binder identification described in this work. First is the SPENSER parallel peptide synthesizer, developed as a part of this diploma project, which can be used for peptide ligand discovery and optimization as well as validation of ligands identified using display techniques. Several libraries consisting of a total of 1 052 peptides have been prepared and then used to describe its potential applications. A sample of 154 preparations, representing 14.6 % analytical coverage of the prepared libraries, showed an average purity of 67 ± 19 % according to LC-MS. The libraries presented illustrate that SPENSER is a suitable tool for the parallel synthesis of linear and disulfide-cyclized peptides with limited variability, or libraries consisting of short...
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Antimicrobial peptides isolated from the venom of hymenopterous insect
Monincová, Lenka ; Čeřovský, Václav (advisor) ; Macek, Tomáš (referee) ; Fusek, Martin (referee)
Rapid development of bacterial resistance and multiresitance to conventional antibiotics has resulted in an intensive search for alternative antimicrobial agents. Antimicrobial peptides (AMPs) belong to promising anti-infective candidates since they do not development bacterial resistance. They kill microbes by disturbing or permeabilizing the cytoplasmic membrane, or may target putative key intracellular compartments. Their advantages include fast action and selectivity between prokaryotic and eukaryotic cells. We have isolated several novel AMPs from the venom of wild bees: halictines (HAL-1 and HAL-2) from Halictus sexcinctus, lasiocepsin (Las) from Lasioglossum laticeps and macropin (MAC-1) from Macropis fulvipes. They are active against Gram-positive and Gram- negative bacteria and against yeast Candida albicans. While halictines and macropin have moderate hemolytic activity, Las shows no hemolytic activity. A novel AMP was isolated also from the mucus of Xiphydria camelus. This AMP belongs to the category of insect defensins. It contains 55 amino acid residues, three disulphide bridges and its C-terminus is amidated. CD and NMR studies of HAL-1, HAL-2 and MAC-1 revealed propensity to form amphipathic α-helical structure in the presence of sodium dodecyl sulfate or trifluoroethanol. For the...
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New analogues of human insulin with covalently stabilized cyclic structures in the C-terminus of the B-chain
Kaplan, Vojtěch ; Stiborová, Marie (advisor) ; Flegel, Martin (referee)
Diabetes mellitus is considered as one of world's most common metabolic diseases. Complicated treatment and increasing number of newly diagnosed patients, suffering from diabetes every year, shows the importance and necessity of research in this area. Some of the major aims of this research are the development of new therapeutically utilized drugs and defining the problems of insulin acting in human body. Insulin is a peptide hormone whose main physiological function is to regulate blood glucose level in organism connected with large impact on whole metabolism. Insulin acts through binding of its monomeric form to the insulin receptor. Upon binding to the receptor molecule of insulin undergoes specific structural changes, which put the hormone into an active state. As of now, the structure of the insulin's active monomeric form is still unknown. By testing binding affinities of many modified insulin analogues there was discovered strong evidence between structural conformation of the C-terminus of the B-chain and binding affinity to the receptor. The most crucial data, necessary for this work, were observed from the structure of highly active insulin analogues that possessed unique B26 turn, recently prepared and described by team of Dr. J. Jiráček, IOCB AS CR. The aim of this work was synthesis of...
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Synthetic scan of C-domain from prion proteins
Šebestík, Jaroslav ; Zawada, Zbigniew ; Šafařík, Martin ; Hlaváček, Jan
Prions are suspected as culprits of several neuropathogenic diseases. A combination of chemical and recombinant syntheses is a powerful tool for studying prion role in pathogenesis of neurodegenerative diseases. Due to the presence of “difficult” sequences in the prion protein molecule, chemical ligation procedure using peptide thioesters as key building blocks is a method of choice. Therefore, a synthetic scan of peptides constituting the mouse prion protein (MoPrP) C-domain 93-231 was carried out. The synthesis on chlorotritylchloride resin was the most promising for most peptides. Only the octadecapeptide MoPrP195-212 could not be synthesized by automated peptide synthesis even using the β-sheet breaking dipeptides. This difficulty was overcome by manual peptide synthesis with careful monitoring of coupling and Fmoc removal.
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Larvae of flesh fly Neobellieria bullata as a source for novel antimicrobial peptides
Neubauerová, Tereza ; Macková, Martina ; Macek, Tomáš ; Šanda, Miloslav ; Voburka, Zdeněk
The antibiotic resistant strains of microorganisms are increasing threat for public health. Antimicrobial peptides from natural sources display a wide spectrum of activity. In our laboratory, several peptide fractions with antimicrobial acivity against gram-positive and gramnegative bacteria and fungi were isolated from larvae of flesh fly Neobellieria bullata. The presence of peptides was proved by tricine electrophoresis, molecular weights and sequences determination by MS and Edman sequencing, respectively. In comparison with database sequences we obtained structures present mainly in insect and amphibian antimicrobial peptides, but total matches were not higher than in 36%. Upon the obtained sequence motifs we designed structures of several synthetic peptides. These were synthetised on the Rink amide MBHA resin. Synthesis was performed according Fmoc/PyBOP-DIEA protocol. Antimicrobial activity of these peptides was determined.
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Different charged short lipopeptides
Ježek, R. ; Slaninová, Jiřina ; Králová, M. ; Macková, M.
Series of short peptides palmitoylated in different positions was designed, synthesized and tested for antimicrobial activities. The palmitoyl group was situated either on the alpha - amino group in position 1 or on the epsilon - amino group of lysine in position 3. The peptide charge ranged from –3 to +4. The peptide part of the lipopeptides constituted mainly from lysine, arginine and glutamic acid.
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Lucifensin, a peptide behind the maggot therapy
Čeřovský, Václav
The larvae of the green bottle fly (Lucilia sericata) are routinely used as a medication for the treatment of necrotic, non-healing and highly infected wound in many hospitals all over the world. We have identified and determined primary structure of the defensin originated from the immune system of Lucilia sericata larvae and named it lucifensin. This is 40 amino acid residues and three intramolecular disulfide bridges peptide. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We studied the importance of lucifensin disulfide bridges and the N-terminal part of its molecule for the structure and for antimicrobial activity.
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Design of stable antimicrobial peptides through hydrocarbon stapling
Chapuis, Hubert Jean ; Slaninová, Jiřina ; Monincová, Lenka ; Bednárová, Lucie ; Čeřovský, Václav
From the venom of wild bee Lasioglossum laticeps we have recently isolated novel antimicrobial peptides named lasioglossins. One of them, LL-III (VNWKKILGKIIKVVK-NH2), is an amphipathic α-helical peptide which shows strong antimicrobial properties and a low hemolytic activity. We anticipated that the incorporation of an all-hydrocarbon staple (bridge) into the LL-III sequence could increase its propensity to form an α-helix and lead to an improvement of its proteolytic stability as well as increase the antimicrobial activity. LL-III analogs featuring olefinic side chains in various positions were prepared by solid phase peptide synthesis. Ring closing olefin metatheses catalyzed by Grubbs-I catalyst were carried out on the solid support, either between i and i+4 positions or between i and i+7 positions.
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