Q39952925
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Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Alzheimerova choroba (AD) je progresivní mozkové onemocnění, které poškozuje a nakonec ničí mozkové buňky, což vede ke ztrátě paměti, myšlení a poškození dalších mozkových funkcí. V současné době je AD jednou z hlavních příčin úmrtí ve vyspělých zemích a jediná z první desítky nemocí, u které nemáme k dispozici prostředky na léčbu, prevenci nebo alespoň k významnému zpomalení progrese. Z těchto důvodů se intenzivně hledají nové terapeutické postupy pro zlepšení přežití a kvality života pacientů s AD a jejich rodin. Náš předchozí výzkum vedl k identifikaci nových selektivních nukleosidových inhibitorů MARK4 kinázy, která je zapojená v rozvoji patologie u AD. V předkládaném projektu navrhujeme komplexní translační výzkum vedoucí k syntéze a ověřování biologické aktivity syntetických inhibitorů MARK4 kinázy v in vitro anebo in vivo podmínkách s využitím zvířecích i lidských modelů AD a identifikaci preklinických kandidátů léčiv proti Alzheimerově chorobě.; Alzheimer disease (AD) is a progressive brain disorder that damages and eventually destroys brain cells, leading to a loss of memory, thinking and other brain functions. Currently, AD is one of the major cause of death in developed countries and the only one of the top ten without a means to prevent, cure or significantly slow its progression. Therefore, the new therapeutic concepts are urgently needed to improve survival and quality of life for AD patients and families. Our previous work resulted in identification of novel selective nucleoside based inhibitors of MARK4 kinase, which has been implicated in development of AD pathology. Here we propose complex translational research leading to synthesis and validation of biological activities of MARK4 inhibitors under both in vitro and in vivo conditions using animal and human models of AD and identification of preclinical candidates for anti-Alzheimer drugs.
- MeSH
- Alzheimerova nemoc farmakoterapie MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nukleosidy terapeutické užití MeSH
- nukleotidy terapeutické užití MeSH
- translační biomedicínský výzkum MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- neurologie
- farmacie a farmakologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
24 s. : il. ; 21 cm
- MeSH
- organická chemie MeSH
- puriny chemická syntéza farmakologie MeSH
- Publikační typ
- vysokoškolské kvalifikační práce MeSH
- Konspekt
- Chemie. Mineralogické vědy
- NLK Obory
- chemie, klinická chemie
- biochemie
We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing cationic substituents (protonated amino, methylamino, dimethylamino and trimethylammonium groups) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through hex-1-ynyl or propargyl linker. These cationic dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase. In primer extension (PEX), we successfully obtained DNA containing one, two, three, or (all) four modified nucleotides, each bearing a different cationic modification. The cationic dNTPs were somewhat worse substrates compared to previously studied dNTPs bearing hydrophobic or anionic modifications, but the polymerase was still able to synthesize sequences up to 73 modified nucleotides. We also successfully combined one cationic modification with one anionic and two hydrophobic modifications in PEX. In polymerase chain reaction (PCR), we observed exponential amplification only in the case of one cationic modification, while the combination of more cationic nucleotides gave either very low amplification or no PCR product. The hypermodified oligonucleotides prepared by PEX were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies of hybridization, denaturation, and circular dichroism spectroscopy showed that the presence of cationic modifications increases the stability of duplexes.
Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.
- MeSH
- DNA-dependentní DNA-polymerasy * genetika MeSH
- messenger RNA genetika MeSH
- nukleotidy chemie MeSH
- RNA * genetika chemie MeSH
- Publikační typ
- časopisecké články MeSH
5-(β-d-Glucopyranosyloxymethyl)-2'-deoxyuridine and -cytidine 5'-O-triphosphates were prepared and used for polymerase-mediated (primer extension or PCR) synthesis of DNA containing glucosylated 5-hydroxymethyluracil (5hmU) or 5-hydroxymethyluracil (5hmC). The presence of any glucosylated pyrimidines fully protected DNA from cleavage by type II restriction endonucleases. On the other hand, while the presence of glucosylated 5hmU completely inhibited transcription by bacterial (Escherichia coli) RNA polymerase, the DNA containing the corresponding glucosylated 5hmC allowed a similar level of transcription as natural DNA. This suggests different roles of these hypermodified bases in the epigenetic regulation of transcription in bacteriophages or kinetoplastid parasites. Consequently, enzymatic glucosylation of 5hmC-containing DNA can be used for tuning of transcription activity.
We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA.
Synthesis of base-modified oligonucleotides and DNA by polymerase incorporations of modified nucleoside triphosphates (dNTPs) is summarized. The crosscoupling synthesis of modified dNTPs, methods of their enzymatic incorporations, as well as their applications in bioanalysis and chemical biology are discussed.
- MeSH
- biochemie metody trendy MeSH
- diagnostické techniky molekulární MeSH
- DNA-dependentní DNA-polymerasy * MeSH
- nukleové kyseliny, nukleosidy a nukleotidy * chemická syntéza MeSH
- pojmy organické chemie MeSH
- polymerázová řetězová reakce MeSH
- techniky syntetické chemie MeSH
- techniky syntézy na pevné fázi * MeSH
- Publikační typ
- práce podpořená grantem MeSH
A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2',3'-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- nádorové buněčné linie MeSH
- protinádorové látky chemie farmakologie MeSH
- purinové nukleosidy chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
