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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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Synthesis of new carborane and metallacarborane bulding blocks appliable in design of biologically active compounds
Nekvinda, Jan ; Grüner, Bohumír (advisor) ; Janoušek, Zbyněk (referee)
Compounds with carboxylic and amidic functions belong to basic structural blocks, which are used for construction of functional molecules in organic, organometallic and also in carborane chemistry. However, considering cobalt bis(dicarbollide)(1-) ion, the synthetic ways to these derivatives have been virtually unknown. A published procedure on lithiation in THF and reaction with CO2 leading to mono- and dicarboxylic acids had failed in our hands. Nevertheless, a detailed revision of the experimental conditions provided finally good yields of mixture of both acids, which could be separated by chromatography and crystallization, and compound of general formulation [(1-HOOC-1,2-C2B9H10)(1',2'-C2B9H10)-3,3'-Co(III)]- and stereoisomeric mixture of [(HOOC)2-(1,2-C2B9H10)2-3,3'-Co(III)]- were characterized for the first time by combination of NMR, MS and HPLC. Also, the carboxylic acid derivatives with methylene and ethylene connectors of the general formula [(1-HOOC-(CH2)n-1,2- C2B9H10)(1',2'-C2B9H10)-3,3'-Co(III)]- were prepared by lithiation of Cs1 in DME at low temperatures followed by reaction with BrCH2COOEt and subsequent hydrolysis of the resulting ester or by oxidation of the respective propylhydroxy derivative. The acids were converted to reactive p-nitrophenyl esters...
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Synthesis of 9-deazaguanine derivatives as potential inhibitors of tRNA-guanine transglycosylase (TGT)
Chylíková, Barbora ; Hocek, Michal (advisor) ; Smrček, Stanislav (referee)
Shigellosis is an acute diarrheal disease caused by enterobacteria of the genus Shigella. Worldwide, these bacteria are responsible for about 1 million deaths out of 165 million cases of the disease yearly, of which children under the age of 5 from developing countries are the most vulnerable group. The enzyme tRNA-guanine transglycolase (TGT for short) has been identified as essential for the effective pathogenesis of Shigella bacteria and therefore becomes a potential target for the selective treatment of shigellosis. This bachelor thesis describes the optimization of the preparation of a key intermediate for the synthesis of potential TGT inhibitors based on 8-aryl-9-deazaguanine structure. The synthesis starts from commercially available 2,6-dichloro-9-deazapurine. The main steps of the synthesis are the hydrolysis at position 6, the nucleophilic aromatic substitution of the chlorine group for the amino group in position 2, further the iodation and the introduction of the 3-thienyl group in position 8 by Suzuki coupling.
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Synthesis of new carborane and metallacarborane bulding blocks appliable in design of biologically active compounds
Nekvinda, Jan ; Grüner, Bohumír (advisor) ; Janoušek, Zbyněk (referee)
Compounds with carboxylic and amidic functions belong to basic structural blocks, which are used for construction of functional molecules in organic, organometallic and also in carborane chemistry. However, considering cobalt bis(dicarbollide)(1-) ion, the synthetic ways to these derivatives have been virtually unknown. A published procedure on lithiation in THF and reaction with CO2 leading to mono- and dicarboxylic acids had failed in our hands. Nevertheless, a detailed revision of the experimental conditions provided finally good yields of mixture of both acids, which could be separated by chromatography and crystallization, and compound of general formulation [(1-HOOC-1,2-C2B9H10)(1',2'-C2B9H10)-3,3'-Co(III)]- and stereoisomeric mixture of [(HOOC)2-(1,2-C2B9H10)2-3,3'-Co(III)]- were characterized for the first time by combination of NMR, MS and HPLC. Also, the carboxylic acid derivatives with methylene and ethylene connectors of the general formula [(1-HOOC-(CH2)n-1,2- C2B9H10)(1',2'-C2B9H10)-3,3'-Co(III)]- were prepared by lithiation of Cs1 in DME at low temperatures followed by reaction with BrCH2COOEt and subsequent hydrolysis of the resulting ester or by oxidation of the respective propylhydroxy derivative. The acids were converted to reactive p-nitrophenyl esters...
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Acyclic nucleoside bis-phosphonates as potent inhibitors of 6-oxopurine phosphoribosyltransferases
Špaček, Petr ; Keough, D. T. ; Vrbková, Silvie ; Slavětínská, Lenka ; Janeba, Zlatko ; Naesens, L. ; Edstein, M. D. ; Chavchich, M. ; Wang, T. H. ; de Jersey, J. ; Guddat, L. W. ; Hocková, Dana
Hypoxanthine-guanin-(xanthine) phosphoribosyltransferase (HG(X)PRT) is critical for the survival of malarial parasites Plasmodium falciparum and Plasmodium vivax. These parasites rely on HG(X)PRT to make 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit HG(X)PRT and thus have an anti-plasmodial activity. Crystal structures of human HGPRT in complex with several ANP-based inhibitors suggested that attachment of the second phosphonate group which could occupy the pyrophosphate binding site may lead to increased affinity of these compounds.
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Acyclic nucleoside bisphosphonates as inhibitors of 6-oxopurine phosphoribosyltransferases: Potential antimalarial and antibacterial agents
Hocková, Dana ; Keough, D. T. ; Špaček, Petr ; Janeba, Zlatko ; Edstein, M. D. ; Chavchich, M. ; Wang, T. H. ; Eng, W. S. ; West, N. P. ; de Jersey, J. ; Guddat, L. W.
Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the human, Plasmodium falciparum, P. vivax, Escherichia coli and Mycobacterium tuberculosis 6-oxopurine phosphoribosyltransferases (PRTases), key enzymes of the purine salvage pathway. Chemical modifications based on the crystal structures of several inhibitors in complex with human HGPRTase have led to the design of new ANPs. These novel compounds contain a second phosphonate group attached to the ANP scaff old. The crystal structures of these inhibitors in complex with human HGPRTase show that they can fill three critical locations in the active site: the binding sites of the purine base, the 5’-phosphate group and pyrophosphate. Prodrugs have been synthesized and have been shown to arrest the growth of P. falciparum in erythrocyte culture. Prodrugs of selected ANPs also inhibit the growth of Mycobacterium tuberculosis in cell-based assays.
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